Chimeric insecticidal proteins

ABSTRACT

IRDIG35563 vegetative insecticidal toxins, polynucleotides encoding such toxins, use of such toxins to control pests, and transgenic plants that produce such toxins are disclosed. The invention includes IRDIG35563 variants, fragments and analogs.

CROSS-REFERENCE TO RELATED APPLICATIONS

This Application is a continuation application of U.S. patentapplication Ser. No. 16/650,300, filed Mar. 24, 2020, which is anational stage patent application of International patent applicationno. PCT/US2018/052788 filed Sep. 26, 2018, which claims the benefit ofpriority to U.S. Provisional Application No. 62/563,228 filed on Sep.26, 2017, the disclosures of each of which are incorporated herein byreference in their entireties.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The official copy of the sequence listing is submitted electronicallyvia EFS-Web as an ASCII formatted sequence listing with a file named“80144_WO_PCT_Sequence_Listing_ST25” created on Sep. 25, 2018, andhaving a size of 17 kilobytes and is filed concurrently with thespecification. The sequence listing contained in this ASCII formatteddocument is part of the specification and is herein incorporated byreference in its entirety.

FIELD OF THE INVENTION

This invention generally relates to the field of molecular biology. Morespecifically the invention concerns new insecticidal protein toxinsdeveloped from vegetative insecticidal protein toxins found in Bacillusthuringiensis and their use to control insects.

BACKGROUND OF INVENTION

Insects and other pests cost farmers billions of dollars annually incrop losses and expense to keep these pests under control. In additionto losses in field crops, insect pests are also a burden to vegetableand fruit growers, to producers of ornamental flowers, and to homegardeners. The losses caused by insect pests in agricultural productionenvironments include decrease in crop yield, reduced crop quality, andincreased harvesting costs.

Insect pests are mainly controlled by intensive applications of chemicalpesticides, which are active through inhibition of insect growth,prevention of insect feeding or reproduction, or cause death. Goodinsect control can thus be reached, but these chemicals can sometimesalso affect other beneficial insects. Another problem resulting from thewide use of chemical pesticides is the appearance of resistant insectpopulations. This has been partially alleviated by various resistancemanagement practices, but there is an increasing need for alternativepest control agents. Biological pest control agents, such as Bacillusthuringiensis (Bt) strains expressing pesticidal toxins likedelta-endotoxins, have also been applied to crop plants withsatisfactory results, offering an alternative or compliment to chemicalpesticides. The genes coding for some of these delta-endotoxins havebeen isolated and their expression in heterologous hosts have been shownto provide another tool for the control of economically important insectpests. In particular, the expression of insecticidal toxins, such asBacillus thuringiensis delta-endotoxins, in transgenic plants haveprovided efficient protection against selected insect pests, andtransgenic plants expressing such toxins have been commercialized,allowing farmers to reduce applications of chemical insect controlagents.

The soil microbe Bacillus thuringiensis is a Gram-positive,spore-forming bacterium characterized by parasporal crystalline proteininclusions. Bacillus thuringiensis continues to be the leading source ofnovel insecticidal proteins for development of plant incorporatedpesticides. In the North American maize insect resistance market,Spodoptera frugiperda (fall armyworm “FAW”), Ostrinia nubialis Hübner(European corn borer “ECB”), and Helicoverpa zea Boddie (corn earworm“CEW”) are key driver pests, although there are other key insect pestsin other geographies (e.g. Helicoverpa armigera (cotton bollworm “CBW”or corn earworm “CEW”)) and additional secondary, but important insectpest species. Bt toxins represent over 90% of the bioinsecticideproducts commercially marketed and essentially the entire source ofgenes for transgenic crops that have been developed to provideresistance to insect feeding. Bt bacteria produce insecticidaldelta-endotoxins including Crystal (Cry), Cytotoxin (Cyt), andVegetative Insecticidal Protein (VIP) toxins, depending on their geneand protein structure. Cry toxins are produced during spore formation asinsoluble crystal proteins. VIP toxins, on the other hand, are producedas soluble proteins during the vegetative stage of Bt bacterial growth.VIP proteins are distinct from Cry proteins in their structure, butshare the property with Cry toxins of being pore formers acting on cellslocated in the insect midgut. The classification of VIP protein waspreviously based on their target insect types. Nomenclature is currentlyemployed that systematically classifies the VIP genes based upon aminoacid sequence homology rather than upon insect specificities. Crickmore,N., Baum, J., Bravo, A., Lereclus, D., Narva, K., Sampson, K., Schnepf,E., Sun, M. and Zeigler, D. R. “Bacillus thuringiensis toxinnomenclature” (2016); http://www.btnomenclature.info/; andhttp://www.lifesci.sussex.ac.uk/home/Neil_Crickmore/Bt/vip.html

The continued use of chemical and biological agents to control insectpests heightens the chance for insects to develop resistance to suchcontrol measures. Also, the high selectivity of biological controlagents often results in only a few specific insect pests beingcontrolled by each agent. Despite the success of ECB-resistanttransgenic corn, the possibility of the development of resistant insectpopulations threatens the long-term durability of Cry proteins in ECBcontrol and creates the need to discover and develop new Cry or othertypes of biological control agents to control ECB and other pests.

There remains a need to discover and develop new and effective pestcontrol agents that provide an economic benefit to farmers and that areenvironmentally acceptable. Particularly needed are control agentstargeted to a wide spectrum of economically important insect pests thatefficiently control insect populations that are, or could become,resistant to existing insect control agents and those with equal to orincreased potency compared to current control agents.

BRIEF SUMMARY OF THE INVENTION

The present invention provides insecticidal chimeric VIP toxins,including the protein toxin designated as IRDIG35563 (SEQ ID NO:2) thatwas constructed based on the first 613 amino acid residues of VIP3Ab1and a C-terminal portion of another VIP toxin, variants of IRDIG35563,nucleic acids encoding these chimeric toxins, methods of controllingpests using the toxins, methods of producing the toxins in transgenichost cells, and transgenic plants that express the toxins. The inventionfurther provides a recombinant nucleic acid construct comprising one ormore heterologous regulatory elements that drive expression of a nucleicacid sequence encoding SEQ ID NO:2 and nucleic acid sequences chosenfrom the group consisting of SEQ ID NO:1, SEQ ID NO:3, and SEQ ID NO:4.In another embodiment, the invention provides nucleic acid constructscomprising a nucleotide sequence that encodes an IRDIG35563 chimericinsect toxin operably linked to one or more regulatory regions such as apromoter that is not derived from Bt and is capable of drivingexpression in a host cell, preferably a plant cell. The inventionincludes an insecticidal chimeric toxin comprising residues 1 to 789 ofSEQ ID NO:2, and plants or plant parts comprising such nucleic acidconstructs. The invention further provides plants or plant parts whereinthe chimeric toxin has insecticidal activity against insects selectedfrom the group consisting of Spodoptera exigua (Beet armyworm, BAW),Spodoptera eridania (Southern armyworm, SAW), Spodoptera frugiperda(Fall armyworm, FAW), Cry1F resistant Spodoptera frugiperda (rFallarmyworm, rFAW), Helicoverpa zea (Corn earworm, CEW), Pseudoplusiaincludens (Soybean looper, SBL), Anticarsia gemmatalis (Velvetbeancaterpillar, VBC), Heliothis virescens (Tobacco budworm, TBW), andHelicoverpa armigera (Cotton bollworm, CBW). Also provided by theinvention is a method for controlling susceptible insects comprisingcontacting the gut of said insect with an effective amount of adisclosed chimeric toxin as well as a method for controlling an insectpest population comprising contacting the gut of individuals of saidpest population with a insecticidally effective amount of a disclosedchimeric toxin. Also provided is a method for producing an insectresistant or insect tolerant plant comprising breeding a non-transgenicplant with a transgenic plant comprising a disclosed DNA constructstably incorporated into the genome of the plant and selecting progenycontain the disclosed nucleic acid construct.

DESCRIPTION OF THE SEQUENCES

SEQ ID NO:1 A chimeric DNA sequence encoding IRDIG35563.

SEQ ID NO:2 The chimeric protein toxin sequence of IRDIG35563.

SEQ ID NO:3 A maize optimized High GC content DNA sequence encodingIRDIG35563.

SEQ ID NO:4 A soybean most preferred codon optimized DNA sequenceencoding IRDIG35563.

DETAILED DESCRIPTION OF THE INVENTION Definitions

As used herein the singular forms “a”, “and”, and “the” include pluralreferents unless the context clearly dictates otherwise. Thus, forexample, reference to “a cell” includes a plurality of such cells andreference to “the protein” includes reference to one or more proteinsand equivalents thereof, and so forth. All technical and scientificterms used herein have the same meaning as commonly understood to one ofordinary skill in the art to which this disclosure belongs unlessclearly indicated otherwise.

“Sufficiently identical” is used herein to refer to an amino acidsequence that has at least about 40%, 45%, 50%, 51%, 52%, 53%, 54%, 55%,56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%,70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%0, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or greater sequence identity compared to a reference sequenceusing one of the alignment programs described herein using standardparameters.

As used herein, the term “protein,” “peptide molecule,” or “polypeptide”includes those molecules that undergo modification, includingpost-translational modifications, such as, but not limited to, disulfidebond formation, glycosylation, phosphorylation or oligomerization.

The terms “amino acid” and “amino acids” refer to all naturallyoccurring L-amino acids.

“Retains insecticidal activity” is used herein to refer to a polypeptidehaving at least about 10%, at least about 30%, at least about 50%, atleast about 70%, 80%, 90%, 95% or higher of the insecticidal activity ofthe full-length IRDIG35563 polypeptide.

“Fragments” or “biologically active portions” include polypeptidefragments comprising amino acid sequences sufficiently identical to anIRDIG35563 polypeptide and have insecticidal activity andpolynucleotides encoding the fragments. “Fragments” or “biologicallyactive portions” of IRDIG35563 polypeptides includes fragmentscomprising amino acid sequences sufficiently identical to the amino acidsequence set forth in IRDIG35563 polypeptides of the disclosure, whereinthe polypeptide has insecticidal activity.

An “isolated” nucleic acid molecule (or DNA) is used herein to refer toa nucleic acid sequence (or DNA) that is no longer in its naturalenvironment and have been placed in a difference environment by the handof man, for example in vitro. A “recombinant” nucleic acid molecule (orDNA) is used herein to refer to a nucleic acid sequence (or DNA) that isin a recombinant bacterial or plant host cell. In some embodiments, an“isolated” or “recombinant” nucleic acid is free of sequences(preferably protein encoding sequences) that naturally flank the nucleicacid (i.e., sequences located at the 5′ and 3′ ends of the nucleic acid)in the genomic DNA of the organism from which the nucleic acid isderived.

“Contiguous nucleotides” is used herein to refer to nucleotide residuesthat are immediately adjacent to one another.

As used herein non-genomic nucleic acid sequence, nucleic acid moleculeor polynucleotide refers to a nucleic acid molecule that has one or morechange in the nucleic acid sequence compared to a native or genomicnucleic acid sequence. In some embodiments, the change to a native orgenomic nucleic acid molecule includes but is not limited to: changes inthe nucleic acid sequence due to the degeneracy of the genetic code;optimization of the nucleic acid sequence for expression in plants;changes in the nucleic acid sequence to introduce at least one aminoacid substitution, insertion, deletion and/or addition compared to thenative or genomic sequence; deletion of one or more upstream ordownstream regulatory regions associated with the genomic nucleic acidsequence; insertion of one or more heterologous upstream or downstreamregulatory regions; deletion of the 5′ and/or 3′ untranslated regionassociated with the genomic nucleic acid sequence; insertion of aheterologous 5′ and/or 3′ untranslated region; and modification of apolyadenylation site. In some embodiments, the non-genomic nucleic acidmolecule is a synthetic nucleic acid sequence.

The present invention provides insecticidal protein toxins and methodsfor delivering the toxins that are functionally active and effectiveagainst many orders of insects, especially Lepidopteran insects. By“functional activity” “functional” “insecticidal” or “active against” itis meant that the protein is functional as an orally active toxin orinsect control agent, that the protein has a toxic effect, or is able todisrupt or deter insect growth or feeding. When an insect ingests aneffective amount of a toxin of the subject invention delivered viatransgenic plant expression, formulated protein composition(s),sprayable protein composition(s), a bait matrix or other deliverysystem, the results are typically death of the insect, inhibition of thegrowth or proliferation of the insect, or prevention of the insects fromfeeding upon the source, preferably a transgenic plant, that makes thetoxins available to the insects. Functional proteins of the subjectinvention can also work together or alone to enhance or improve theactivity of one or more other toxin proteins. The terms “toxic,”“toxicity,” or “toxin” are meant to convey that the subject toxins havefunctional activity as defined herein.

Complete lethality to feeding insects is preferred but is not requiredto achieve functional activity. If an insect avoids the toxin or ceasesfeeding, that avoidance will be useful in some applications, even if theeffects are sublethal or lethality is delayed or indirect. For example,if insect resistant transgenic plants are desired, the reluctance ofinsects to feed on the plants is as useful as lethal toxicity to theinsects because the ultimate objective is avoiding insect-induced plantdamage.

Using various strains of bacterial isolates, we have invented newchimeric toxins that are active against an unexpectedly large array ofcommercially important insect pests. We describe here the invention ofnew chimeric VIP toxins that have unexpectedly broad spectruminsecticidal activity, including insecticidal activity againstSpodoptera exigua (Beet armyworm, BAW), Spodoptera eridania (Southernarmyworm, SAW), Spodoptera frugiperda (Fall armyworm, FAW) and FAWresistant to deregulated plant incorporated protectants, Helicoverpa zea(Corn earworm, CEW), Pseudoplusia includens (Soybean looper, SBL),Anticarsia gemmatalis (Velvetbean caterpillar, VBC), Heliothis virescens(Tobacco budworm, TBW), and Helicoverpa armigera (Cotton bollworm, CBW).

A class of insecticidal proteins called VIP3 has been described. See,e.g., Estruch et al. (1996, Proc. Natl. Acad. Sci. 93:5389-5394) and Yuet al. (1997, Appl. Environ. Microbiol. 63:532-536). VIP3 genes encodeapproximately 88 kDa proteins that are expressed by Bts duringvegetative stages of growth. These toxins were reported to be distinctfrom crystal-forming delta-endotoxins. Reference to VIP toxinsdesignated VIP1A(a), VIP1A(b), VIP2A(a), VIP2A(b), VIP3A(a), andVIP3A(b) is known in the literature. See also Lee et al., AEM vol. 69,no. 8 (August 2003), pages 4648-4657, for a discussion of the mechanismof action and truncation of VIP proteins.

VIP3A protein toxins possess insecticidal activity against manylepidopteran pests, including FAW, CEW, Agrotis ipsilon Hufnagel (blackcutworm “BCW”), and Heliothis virescens Fabricius (tobacco budworm“TBW”). More recently, VIP proteins have been found to be toxic tocertain species of hemipteran insect pests (Nanasaheb, P. et al, Toxins(Basel) vol. 4, no. 6 (June 2012), pages 405-429, Sattar S. and Maiti M.K., J. Microbiol. Biotechnol. 2011, 21:937-946). Thus, the VIP class ofprotein toxins display a unique spectrum of insecticidal activities.Other disclosures, WO 98/18932, WO 99/33991, WO 98/00546, and WO99/57282, have also now identified homologues of the VIP3 class ofproteins.

IRDIG35563 polypeptides and variants and fragments there. A novelVIP-based chimeric insecticidal protein, IRDIG35563, was created bycombining the N-terminal 613 amino acids of VIP3Ab1 with the C-terminal176 amino acids of IRDIG10870 (aka DIG740). Because of the redundancy ofthe genetic code, a variety of different nucleic acid sequences canencode the amino acid sequences disclosed herein. It is well within theskill of a person trained in the art to create these alternative nucleicacid sequences encoding the same, or essentially the same, toxins oncethe amino acid sequences are known.

Insect active variants of the IRDIG35563 toxin are also describedherein, and are referred to collectively as IRDIG35563 insect toxins, orIRDIG35563 variants, and include truncated forms having N-terminal Metresidues added to N-terminal truncations. IRDIG35563 variants aredefined based on SEQ ID NO:2. For full length toxins, conservative aminoacid substitutions varying no more than 5% from SEQ ID NO:2 that retainactivity are within the scope of this invention.

In some embodiments, the IRDIG35563 polypeptide has at least about 40%,45%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%,63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%,77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identitycompared to SEQ ID NO: 2, as well as amino acid substitutions,deletions, insertions, fragments thereof, and combinations thereof. Theterm “about” when used herein in context with percent sequence identitymeans+/−0.5%. These values can be appropriately adjusted to determinecorresponding homology of proteins considering amino acid similarity andthe like.

In some embodiments, the sequence identity is against the full-lengthsequence of an IRDIG35563 polypeptide.

In some embodiments fragments of IRDIG35563 polypeptides comprise anamino acid sequence sufficiently identical to the amino acid sequenceset forth in IRDIG35563 polypeptides of the disclosure, wherein thepolypeptide has insecticidal activity. Such biologically active portionscan be prepared by recombinant techniques and evaluated for insecticidalactivity. In some embodiments, the IRDIG35563 polypeptide retainsinsecticidal activity against a Lepidopteran species. In someembodiments, the insecticidal activity is against one or more insectpests selected from BAW, SAW, FAW, rFAW, CEW, TBW, SBL, VBC, and CBW.

In some embodiments, the polypeptide fragment is an N-terminal and/or aC-terminal truncation of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31 or more amino acids from the N-terminus and/or C-terminus, byproteolysis, or by insertion of a start codon, by deletion of the codonsencoding the deleted amino acids and concomitant insertion of a startcodon, and/or insertion of a stop codon in the sequence encoding thepolypeptide fragment.

In some embodiments, the IRDIG35563 polypeptide fragment is anN-terminal truncation of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 amino acids from theN-terminus of the IRDIG35563 polypeptide of SEQ ID NO: 2.

In some embodiments, the IRDIG35563 polypeptide fragment is anN-terminal and/or a C-terminal truncation of at least 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34 or more amino acids from theN-terminus and/or C-terminus relative to the IRDIG35563 polypeptide ofSEQ ID NO: 2.

In some embodiments, an IRDIG35563 polypeptide comprises an amino acidsequence having at least about 40%, 45%, 50%, 51%, 52%, 53%, 54%, 55%,56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%,70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or greater identity to the amino acid sequence of theIRDIG35563 polypeptide of SEQ ID NO: 2, wherein the IRDIG35563polypeptide has insecticidal activity.

A surprising property of IRDIG35563 is that it was found to be activeagainst populations of FAW that have become resistant to certain Crytoxins, especially Cry1F. Accordingly, IRDIG35563 toxins are idealcandidates for control and prevention of resistant Lepidopteran pestpopulations. Another surprising property of IRDIG35563 toxins are theirbroad spectrum of activity against many Lepidopteran crop pests. Anothersurprising property of these chimeric toxins are their activity againstLepidopteran species that are pests of both maize and soybean includingBAW, SAW, FAW, rFAW, CEW, TBW, SBL, VBC, and CBW.

IRDIG35563 toxins and insecticidally active variants. In addition to thefull length IRDIG35563 toxin of SEQ ID NO:2, the invention encompassesinsecticidally active variants of SEQ ID NO:2. By the term variant,applicants intend to include certain deletion, substitution, andinsertion mutants. An IRDIG35563 fragment is any protein sequence thatis found in SEQ ID NO:2 that is less than the full length IRDIG35563amino acid sequence and which has insecticidal properties. IRDIG35563variant fragments are also contemplated as part of the invention and aredefined as fragments of IRDIG35563 containing certain deletion,substitution, and insertion mutants described herein and which haveinsecticidal activity.

IRDIG35563 variants created by making a limited number of amino aciddeletions, substitutions, or additions. Amino acid deletions,substitutions, and additions to the amino acid sequence of SEQ ID NO:2can readily be made in a sequential manner and the effects of suchvariations on insecticidal activity can be tested by bioassay. Theinvention also includes insecticidally active variants of SEQ ID NO:2,in which up to 39 conservative independent amino substitutions have beenmade.

Amino acid sequence variants of an IRDIG35563 polypeptide can beprepared by mutations in the DNA. This may also be accomplished by oneof several forms of mutagenesis and/or in directed evolution.

In some aspects, the changes encoded in the amino acid sequence will notsubstantially affect the function of the protein. Such variants willpossess the desired pesticidal activity. However, it is understood thatthe ability of an IRDIG35563 polypeptide to confer pesticidal activitymay be improved using such techniques upon the compositions of thisdisclosure.

In some embodiments, the translation initiator methionine of theIRDIG35563 polypeptide is cleaved off post translationally, for exampleby a methionine aminopeptidase in many cellular expression systems.

Variants may be made by making random mutations or the variants may bedesigned. In the case of designed mutants, there is a high probabilityof generating variants with similar activity to the native toxin whenamino acid identity is maintained in critical regions of the toxin whichaccount for biological activity or are involved in the determination ofthree-dimensional configuration which ultimately is responsible for thebiological activity. A high probability of retaining activity will alsooccur if substitutions are conservative. Amino acids may be placed inthe following classes: non-polar, uncharged polar, basic, and acidic.Conservative substitutions whereby an amino acid of one class isreplaced with another amino acid of the same type are least likely tomaterially alter the biological activity of the variant. Table 1provides a listing of examples of amino acids belonging to each class.

TABLE 1 Classes of amino acids Class of Amino Acid Examples of AminoAcids Nonpolar Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Side ChainsMet (M), Phe (F), Trp (W) Uncharged Polar Gly (G), Ser (S), Thr (T), Cys(C), Tyr (Y), Side Chains Asn (N), Gln (Q) Acidic Side Chains Asp (D),Glu (E) Basic Side Chains Lys (K), Arg (R), His (H) Beta-branched SideChains Thr, Val, Ile Aromatic Side Chains Tyr, Phe, Trp, His

Alternatively, alterations may be made to the protein sequence of manyproteins at the amino or carboxy terminus without substantiallyaffecting activity. This can include insertions, deletions oralterations introduced by modern molecular methods, such as PCR,including PCR amplifications that alter or extend the protein codingsequence by inclusion of amino acid encoding sequences in theoligonucleotides utilized in the PCR amplification. Alternatively, theprotein sequences added can include entire protein-coding sequences, togenerate protein fusions. Such fusion proteins are often used to (1)increase expression of a protein of interest (2) introduce a bindingdomain, enzymatic activity or epitope to facilitate either proteinpurification, protein detection or other experimental uses (3) targetsecretion or translation of a protein to a subcellular organelle, suchas the periplasmic space of Gram-negative bacteria, mitochondria orchloroplasts of plants or the endoplasmic reticulum of eukaryotic cells,the latter of which often results in glycosylation of the protein.

Variant nucleotide and amino acid sequences of the disclosure alsoencompass sequences derived from mutagenic and recombinogenic proceduressuch as DNA shuffling. With such a procedure, one or more differentIRDIG35563 polypeptide coding regions can be used to create a newIRDIG35563 polypeptide possessing the desired properties. In thismanner, libraries of recombinant polynucleotides are generated from apopulation of related sequence polynucleotides comprising sequenceregions that have substantial sequence identity and can be homologouslyrecombined in vitro or in vivo. For example, using this approach,sequence motifs encoding a domain of interest may be shuffled between apesticidal gene and other known pesticidal genes to obtain a new genecoding for a protein with an improved property of interest, such as anincreased insecticidal activity. Strategies for such DNA shufflinginclude for example, Stemmer, (1994) Proc. Natl. Acad. Sci. USA91:10747-10751; Stemmer, (1994) Nature 370:389-391; Crameri, et al.,(1997) Nature Biotech. 15:436-438; Moore, et al., (1997) J. Mol. Biol.272:336-347; Zhang, et al., (1997) Proc. Natl. Acad. Sci. USA94:4504-4509; Crameri, et al., (1998) Nature 391:288-291; and U.S. Pat.Nos. 5,605,793 and 5,837,458.

Domain swapping or shuffling is another mechanism for generating alteredIRDIG35563 polypeptides. Domains may be swapped between IRDIG35563polypeptides resulting in further hybrid or chimeric toxins withimproved insecticidal activity or target spectrum.

Pesticidal proteins of the present invention are encoded by a nucleotidesequence sufficiently identical to the nucleotide sequence of SEQ IDNO:1, or the pesticidal proteins are sufficiently identical to the aminoacid sequence set forth in SEQ ID NO:2.

To determine the percent identity of two amino acid sequences or of twonucleic acids, the sequences are aligned for optimal comparisonpurposes. The percent identity between the two sequences is a functionof the number of identical positions shared by the sequences (i.e.,percent identity=number of identical positions/total number of positions(e.g., overlapping positions)×100). In one embodiment, the two sequencesare the same length. In another embodiment, the percent identity iscalculated across the entirety of the reference sequence. The percentidentity between two sequences can be determined using techniquessimilar to those described below, with or without allowing gaps. Incalculating percent identity, typically exact matches are counted. Agap, (a position in an alignment where a residue is present in onesequence but not in the other) is regarded as a position withnon-identical residues.

The determination of percent identity between two sequences can beaccomplished using a mathematical algorithm. A non-limiting example of amathematical algorithm utilized for the comparison of two sequences isthe algorithm incorporated into the BLASTN and BLASTX programs. Karlinand Altschul (1990) Proc. Nat'l. Acad. Sci. USA 87:2264, Altschul et al.(1990) J. Mol. Bioi. 215:403, and Karlin and Altschul (1993) Proc.Nat'l. Acad. Sci. USA 90:5873-5877. BLAST nucleotide searches can beperformed with the BLASTN program, score=100, word length=12, to obtainnucleotide sequences homologous to pesticidal-like nucleic acidmolecules of the invention. BLAST protein searches can be performed withthe BLASTX program, score=50, word length=3, to obtain amino acidsequences homologous to pesticidal protein molecules of the invention.To obtain gapped alignments for comparison purposes, Gapped BLAST (inBLAST 2.0) can be utilized as described in Altschul et al. (1997)Nucleic Acids Res. 25:3389. Alternatively, PSI-Blast can be used toperform an iterated search that detects distant relationships betweenmolecules. See Altschul et al. (1997) supra. When utilizing BLAST,Gapped BLAST, and PSI-Blast programs, the default parameters of therespective programs (e.g., BLASTX and BLASTN) can be used. Alignment mayalso be performed manually by inspection.

Another non-limiting example of a mathematical algorithm utilized forthe comparison of sequences is the ClustalW algorithm (Higgins et al.(1994) Nucleic Acids Res. 22:4673-4680). ClustalW compares sequences andaligns the entirety of the amino acid or DNA sequence, and thus canprovide data about the sequence conservation of the entire amino acidsequence. The ClustalW algorithm is used in several commerciallyavailable DNA/amino acid analysis software packages, such as the ALIGNXmodule of the Vector NTI Program Suite (Invitrogen Corporation,Carlsbad, Calif.). After alignment of amino acid sequences withClustalW, the percent amino acid identity can be assessed. Anon-limiting example of a software program useful for analysis ofClustalW alignments is GENEDOC™ GENEDOC™ (Karl Nicholas) allowsassessment of amino acid (or DNA) similarity and identity betweenmultiple proteins. Another non-limiting example of a mathematicalalgorithm utilized for the comparison of sequences is the algorithm ofMyers and Miller (1988) CABIOS 4(1):11-17. Such an algorithm isincorporated into the ALIGN program (version 2.0), which is part of theGCG Wisconsin Genetics Software Package, Version 10 (available fromAccelrys, Inc., San Diego, Calif., USA). When utilizing the ALIGNprogram for comparing amino acid sequences, a PAM120 weight residuetable, a gap length penalty of 12, and a gap penalty of 4 can be used.Unless otherwise stated, GAP Version 10, which uses the algorithm ofNeedleman and Wunsch (1970) J. Mol. Biol. 48(3):443-453, will be used todetermine sequence identity or similarity using the followingparameters: % identity and % similarity for a nucleotide sequence usingGAP Weight of 50 and Length Weight of 3, and the nwsgapdna.cmp scoringmatrix; % identity or % similarity for an amino acid sequence using GAPweight of 8 and length weight of 2, and the BLOSUM62 scoring program.Equivalent programs may also be used. By “equivalent program” isintended any sequence comparison program that, for any two sequences inquestion, generates an alignment having identical nucleotide residuematches and an identical percent sequence identity when compared to thecorresponding alignment generated by GAP Version 10.

In another embodiment of the invention, protease cleavage sites may beengineered at desired locations to affect protein processing within themidgut of susceptible larvae of certain insect pests. These proteasecleavage sites may be introduced by methods such as chemical genesynthesis or splice overlap PCR (Horton et al., 1989). Serine proteaserecognition sequences, for example, can optionally be inserted atspecific sites in the Cry protein structure to affect protein processingat desired deletion points within the midgut of susceptible larvae.Serine proteases that can be exploited in such fashion includeLepidopteran midgut serine proteases such as trypsin or trypsin-likeenzymes, chymotrypsin, elastase, etc. (Christeller et al., 1992).Further, deletion sites identified empirically by sequencing Cry proteindigestion products generated with unfractionated larval midgut proteasepreparations or by binding to brush border membrane vesicles can beengineered to effect protein activation.

Lepidopteran and Coleopteran serine proteases such as trypsin,chymotrypsin and cathepsin G-like protease, Lepidopteran and Coleopterancysteine proteases such as cathepsins (B-like, L-like, O-like, andK-like proteases) (Koiwa et al., (2000) and Bown et al., (2004)),Lepidopteran and Coleopteran metalloproteases such as ADAM10(Ochoa-Campuzano et al., (2007)), and Lepidoperan and Coleopteranaspartic acid proteases such as cathepsins D-like and E-like, pepsin,plasmin, and chymosin may further be exploited by engineeringappropriate recognition sequences at desired processing sites to affectCry protein processing within the midgut of susceptible larvae ofcertain insect pests and perhaps also function to provide activityagainst non-susceptible insect pests.

IRDIG35563 variants can be produced by introduction or elimination ofprotease processing sites at appropriate positions in the codingsequence to allow, or eliminate, proteolytic cleavage of a largervariant protein by insect, plant, or microorganism proteases are withinthe scope of the invention. The end result of such manipulation isunderstood to be the generation of toxin fragment molecules having thesame or better function and/or activity as the intact (full length)toxin protein.

In another embodiment, fusion proteins are provided that include withinits amino acid sequence an amino acid sequence comprising an IRDIG35563polypeptide of the disclosure. Polynucleotides encoding an IRDIG35563polypeptide may be fused to signal sequences which will direct thelocalization of the IRDIG35563 polypeptide to particular compartments ofa prokaryotic or eukaryotic cell and/or direct the secretion of theIRDIG35563 polypeptide of the embodiments from a prokaryotic oreukaryotic cell. For example, in E. coli, one may wish to direct theexpression of the protein to the periplasmic space. Examples of signalsequences or proteins (or fragments thereof) to which the IRDIG35563polypeptide may be fused to direct the expression of the polypeptide tothe periplasmic space of bacteria include, but are not limited to, thepelB signal sequence, the maltose binding protein (MBP) signal sequence,MBP, the ompA signal sequence, the signal sequence of the periplasmic E.coli heat-labile enterotoxin B-subunit and the signal sequence ofalkaline phosphatase. Several vectors are commercially available for theconstruction of fusion proteins which will direct the localization of aprotein, such as the pMAL series of vectors (particularly the pMAL-pseries) available from New England Biolabs. In a specific embodiment,the IRDIG35563 polypeptide may be fused to the pelB pectate lyase signalsequence to increase the efficiency of expression and purification ofsuch polypeptides in Gram-negative bacteria (see, U.S. Pat. Nos.5,576,195 and 5,846,818). The fusion protein may be a plant plastidtransit peptide/polypeptide fusions or an Apoplast transit peptides suchas rice or barley alpha-amylase secretion signal. The plastid transitpeptide is generally fused N-terminal to the polypeptide to be targeted(e.g., the fusion partner). In one embodiment, the fusion proteinconsists essentially of the plastid transit peptide and the IRDIG35563polypeptide to be targeted. In another embodiment, the fusion proteincomprises the plastid transit peptide and the polypeptide to betargeted. In such embodiments, the plastid transit peptide is preferablyat the N-terminus of the fusion protein. However, additional amino acidresidues may be N-terminal to the plastid transit peptide if the fusionprotein is at least partially targeted to a plastid. In a specificembodiment, the plastid transit peptide is in the N-terminal half,N-terminal third or N-terminal quarter of the fusion protein. Most orall of the plastid transit peptide is generally cleaved from the fusionprotein upon insertion into the plastid. The position of cleavage mayvary slightly between plant species, at different plant developmentalstages, because of specific intercellular conditions or the combinationof transit peptide/fusion partner used. In one embodiment, the plastidtransit peptide cleavage is homogenous such that the cleavage site isidentical in a population of fusion proteins. In another embodiment, theplastid transit peptide is not homogenous, such that the cleavage sitevaries by 1-10 amino acids in a population of fusion proteins. Theplastid transit peptide can be recombinantly fused to a second proteinin one of several ways. For example, a restriction endonucleaserecognition site can be introduced into the nucleotide sequence of thetransit peptide at a position corresponding to its C-terminal end andthe same or a compatible site can be engineered into the nucleotidesequence of the protein to be targeted at its N-terminal end. Care mustbe taken in designing these sites to ensure that the coding sequences ofthe transit peptide and the second protein are kept “in frame” to allowthe synthesis of the desired fusion protein. In some cases, it may bepreferable to remove the initiator methionine of the second protein whenthe new restriction site is introduced. The introduction of restrictionendonuclease recognition sites on both parent molecules and theirsubsequent joining through recombinant DNA techniques may result in theaddition of one or more extra amino acids between the transit peptideand the second protein. This generally does not affect targetingactivity if the transit peptide cleavage site remains accessible and thefunction of the second protein is not altered by the addition of theseextra amino acids at its N-terminus. Precise cleavage site between thetransit peptide and the second protein (with or without its initiatormethionine) can be created using gene synthesis (Stemmer, et al., (1995)Gene 164:49-53) or similar methods. In addition, the transit peptidefusion can intentionally include amino acids downstream of the cleavagesite. The amino acids at the N-terminus of the mature protein can affectthe ability of the transit peptide to target proteins to plastids and/orthe efficiency of cleavage following protein import. This may bedependent on the protein to be targeted. See, e.g., Comai, et al.,(1988) J. Biol. Chem. 263(29):15104-9. In some embodiments, theIRDIG35563 polypeptide is fused to a heterologous signal peptide orheterologous transit peptide.

Nucleic acid molecules, and variants and fragments thereof. Isolated orrecombinant nucleic acid molecules comprising nucleic acid sequencesencoding IRDIG35563 polypeptides or biologically active portionsthereof, as well as nucleic acid molecules sufficient for use ashybridization probes to identify nucleic acid molecules encodingproteins with regions of sequence homology are provided. As used herein,the term “nucleic acid molecule” refers to DNA molecules (e.g.,recombinant DNA, cDNA, genomic DNA, plastid DNA, mitochondrial DNA) andRNA molecules (e.g., mRNA) and analogs of the DNA or RNA generated usingnucleotide analogs. The nucleic acid molecule can be single-stranded ordouble-stranded, but preferably is double-stranded DNA.

Nucleotide sequences that encode IRDIG35563, its variants andtruncations, may be synthesized and cloned into standard plasmid vectorsby conventional means, or may be obtained by standard molecular biologymanipulation of other constructs containing the nucleotide sequences.Unique restriction sites internal to an IRDIG35563 coding region may beidentified and DNA fragments comprising the sequences between therestriction sites of the IRDIG35563 coding region may be synthesized,each such fragment encoding a specific deletion, insertion or otherIRDIG35563 variation. The DNA fragments encoding the modified IRDIG35563fragments may be joined to other IRDIG35563 coding region fragments orother Cry or VIP coding region fragments at appropriate restrictionsites to obtain a coding region encoding the desired full-lengthIRDIG35563 protein, deleted or variant IRDIG35563 protein. For example,one may identify an appropriate restriction recognition site at thestart of a first IRDIG35563 coding region, and a second restriction siteinternal to the IRDIG35563 coding region. Cleavage of this firstIRDIG35563 coding region at these restriction sites would generate a DNAfragment comprising part of the first IRDIG35563 coding region. A secondDNA fragment flanked by analogously-situated compatible restrictionsites specific for another IRDIG35563 coding region may be used incombination with the first DNA restriction fragment to construct avariant.

In some embodiments, the nucleic acid molecule encoding an IRDIG35563polypeptide is a polynucleotide having the sequence set forth in SEQ IDNO: 1, and variants, fragments and complements thereof. Nucleic acidsequences that are complementary to a nucleic acid sequence of theembodiments or that hybridize to a sequence of the embodiments are alsoencompassed. The nucleic acid sequences can be used in DNA constructs orexpression cassettes for transformation and expression in organisms,including microorganisms and plants. The nucleotide or amino acidsequences may be synthetic sequences that have been designed forexpression in an organism including, but not limited to, a microorganismor a plant.

In some embodiments, the nucleic acid molecule encoding the IRDIG35563polypeptide is a non-genomic nucleic acid sequence.

In some embodiments, the nucleic acid molecule encoding an IRDIG35563polypeptide is a non-genomic polynucleotide having a nucleotide sequencehaving at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%,61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%,75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greateridentity, to the nucleic acid sequence of SEQ ID NO: 1, wherein theencoded IRDIG35563 polypeptide has insecticidal activity.

In some embodiments, the IRDIG35563 polynucleotide encodes an IRDIG35563polypeptide having at least about 40%, 45%, 50%, 51%, 52%, 53%, 54%,55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%,69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or greater sequence identity compared to SEQ ID NO: 2, andhas at least one amino acid substitution, deletion, insertion orcombination therefore, compared to the native sequence.

In some embodiments, the nucleic acid molecule encodes an IRDIG35563polypeptide comprising an amino acid sequence having at least about 75%,76%, 77%, 78%, 79%, 80%8, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater identityacross the entire length of the amino acid sequence of SEQ ID NO: 2.

Bacterial genes quite often possess multiple methionine initiationcodons in proximity to the start of the open reading frame. Often,translation initiation at one or more of these start codons will lead togeneration of a functional protein. Start codons can be ATG codons.Certain bacteria such as Bacillus sp. also recognize the codon GTG as astart codon. On rare occasions, translation in bacterial systems caninitiate at a TTG codon. Furthermore, it is not often determined apriori which of these codons are used naturally in the bacterium. Thus,it is understood that use of one of the alternate methionine codons mayalso lead to generation of pesticidal proteins. These pesticidalproteins are encompassed in the present disclosure and may be used inthe methods of the present disclosure. It will be understood that, whenexpressed in plants, it will be necessary to alter the alternate startcodon to ATG for proper translation.

The polynucleotide coding sequence can be modified to add a codon at thepenultimate position following the methionine start codon to create arestriction enzyme site for recombinant cloning purposes and/or forexpression purposes. In some embodiments, the IRDIG35563 polypeptidefurther comprises an alanine residue at the position after thetranslation initiator methionine.

Nucleic acid molecules that are fragments of these nucleic acidsequences encoding IRDIG35563 polypeptides are also encompassed by theembodiments. A fragment of a nucleic acid sequence may encode abiologically active portion of an IRDIG35563 polypeptide or it may be afragment that can be used as a hybridization probe or PCR primer usingmethods disclosed below. Nucleic acid molecules that are fragments of anucleic acid sequence that are contiguous or up to the number ofnucleotides present in a full-length nucleic acid sequence disclosedherein, depending upon the intended use. Fragments of the nucleic acidsequences of the embodiments will encode protein fragments that retainthe biological activity of the IRDIG35563 polypeptide and, hence, retaininsecticidal activity. In some embodiments, the IRDIG35563 polypeptideretains at least about 10%, at least about 30%, at least about 50%, atleast about 70%, 80%, 90%, 95% or higher of the insecticidal activity ofthe full-length IRDIG35563 polypeptide. In some embodiments, theinsecticidal activity is against a Lepidopteran species. In someembodiments, the insecticidal activity is against one or more insectpests selected from BAW, SAW, FAW, CEW, TBW, SBL, VBC, and CBW.

In some embodiments, the nucleic acid encodes an IRDIG35563 polypeptidehaving at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or greater sequence identity compared to SEQ ID NO: 2. In someembodiments, the sequence identity is calculated using ClustalWalgorithm in the ALIGNX® module of the Vector NTI® Program Suite(Invitrogen Corporation, Carlsbad, Calif.) with all default parameters.In some embodiments, the sequence identity is across the entire lengthof polypeptide calculated using ClustalW algorithm in the ALIGNX moduleof the Vector NTI Program Suite (Invitrogen Corporation, Carlsbad,Calif.) with all default parameters.

The embodiments also encompass nucleic acid molecules encodingIRDIG35563 polypeptide variants. “Variants” of the IRDIG35563polypeptide encoding nucleic acid sequences include those sequences thatencode the IRDIG35563 polypeptides disclosed herein but that differconservatively because of the degeneracy of the genetic code as well asthose that are sufficiently identical as discussed above. Variantnucleic acid sequences also include synthetically derived nucleic acidsequences that have been generated, for example, by using site-directedmutagenesis but which still encode the IRDIG35563 polypeptides disclosedas discussed below.

The present disclosure provides isolated or recombinant polynucleotidesthat encode any of the IRDIG35563 polypeptides disclosed herein. Due tothe degeneracy of the genetic code, a multitude of nucleotide sequencesencoding IRDIG35563 polypeptides of the present disclosure exist.

Changes can be introduced by mutation of the nucleic acid sequencesthereby leading to changes in the amino acid sequence of the encodedIRDIG35563 polypeptides, without compromising the functional activity ofthe proteins. Thus, variant nucleic acid molecules can be created byintroducing one or more nucleotide substitutions, additions and/ordeletions into the corresponding nucleic acid sequence disclosed herein,such that one or more amino acid substitutions, additions or deletionsare introduced into the encoded protein. Mutations can be introduced bystandard techniques, such as site-directed mutagenesis and PCR-mediatedmutagenesis. Such variant nucleic acid sequences are also encompassed bythe present disclosure.

Alternatively, variant nucleic acid sequences can be made by introducingmutations randomly along all or part of the coding sequence, such as bysaturation mutagenesis, and the resultant mutants can be screened forability to confer pesticidal activity to identify mutants that retainactivity. Following mutagenesis, the encoded protein can be expressedrecombinantly, and the activity of the protein can be determined usingstandard assay techniques.

The polynucleotides of the disclosure and fragments thereof areoptionally used as substrates for a variety of recombination andrecursive recombination reactions, in addition to standard cloningmethods as set forth in, e.g., Ausubel, Berger and Sambrook, i.e., toproduce additional pesticidal polypeptide homologues and fragmentsthereof with desired properties. A variety of such reactions are known.Methods for producing a variant of any nucleic acid listed hereincomprising recursively recombining such polynucleotide with a second (ormore) polynucleotide, thus forming a library of variant polynucleotidesare also embodiments of the disclosure, as are the libraries produced,the cells comprising the libraries and any recombinant polynucleotideproduced by such methods. Additionally, such methods optionally compriseselecting a variant polynucleotide from such libraries based onpesticidal activity, as is wherein such recursive recombination is donein vitro or in vivo.

A variety of diversity generating protocols, including nucleic acidrecursive recombination protocols are available and fully described inthe art. The procedures can be used separately, and/or in combination toproduce one or more variants of a nucleic acid or set of nucleic acids,as well as variants of encoded proteins. Individually and collectively,these procedures provide robust, widely applicable ways of generatingdiversified nucleic acids and sets of nucleic acids (including, e.g.,nucleic acid libraries) useful, e.g., for the engineering or rapidevolution of nucleic acids, proteins, pathways, cells and/or organismswith new and/or improved characteristics.

While distinctions and classifications are made during the ensuingdiscussion for clarity, it will be appreciated that the techniques areoften not mutually exclusive. Indeed, the various methods can be usedsingly or in combination, in parallel or in series, to access diversesequence variants.

The result of any of the diversity generating procedures describedherein can be the generation of one or more nucleic acids, which can beselected or screened for nucleic acids with or which confer desirableproperties or that encode proteins with or which confer desirableproperties. Following diversification by one or more of the methodsherein or otherwise available to one of skill, any nucleic acids thatare produced can be selected for a desired activity or property, e.g.pesticidal activity or, such activity at a desired pH, etc. This caninclude identifying any activity that can be detected, for example, inan automated or automatable format, by any of the assays in the art,see, e.g., discussion of screening of insecticidal activity, infra. Avariety of related (or even unrelated) properties can be evaluated, inserial or in parallel, at the discretion of the practitioner.

Descriptions of a variety of diversity generating procedures forgenerating modified nucleic acid sequences, e.g., those coding forpolypeptides having pesticidal activity or fragments thereof, are foundin the following publications and the references cited therein: Soong,et al., (2000) Nat Genet 25(4):436-439; Stemmer, et al., (1999) TumorTargeting 4:1-4; Ness, et al., (1999) Nat Biotechnol 17:893-896; Chang,et al., (1999) Nat Biotechnol 17:793-797; Minshull and Stemmer, (1999)Curr Opin Chem Biol 3:284-290; Christians, et al., (1999) Nat Biotechnol17:259-264; Crameri, et al., (1998) Nature 391:288-291; Crameri, et al.,(1997) Nat Biotechnol 15:436-438; Zhang, et al., (1997) PNAS USA94:4504-4509; Patten, et al., (1997) Curr Opin Biotechnol 8:724-733;Crameri, et al., (1996) Nat Med 2:100-103; Crameri, et al., (1996) NatBiotechnol 14:315-319; Gates, et al., (1996) J Mol Biol 255:373-386;Stemmer, (1996) “Sexual PCR and Assembly PCR” In: The Encyclopedia ofMolecular Biology. VCH Publishers, New York. pp. 447-457; Crameri andStemmer, (1995) BioTechniques 18:194-195; Stemmer, et al., (1995) Gene,164:49-53; Stemmer, (1995) Science 270: 1510; Stemmer, (1995) BioTechnology 13:549-553; Stemmer, (1994) Nature 370:389-391 and Stemmer,(1994) PNAS USA 91:10747-10751.

Mutational methods of generating diversity include, for example,site-directed mutagenesis (Ling, et al., (1997) Anal Biochem254(2):157-178; Dale, et al., (1996) Methods Mol Biol 57:369-374; Smith,(1985) Ann Rev Genet 19:423-462; Botstein and Shortle, (1985) Science229:1193-1201; Carter, (1986) Biochem J 237:1-7 and Kunkel, (1987) “Theefficiency of oligonucleotide directed mutagenesis” in Nucleic Acids &Molecular Biology (Eckstein and Lilley, eds., Springer Verlag, Berlin));mutagenesis using uracil containing templates (Kunkel, (1985) PNAS USA82:488-492; Kunkel, et al., (1987) Methods Enzymol 154:367-382 and Bass,et al., (1988) Science 242:240-245); oligonucleotide-directedmutagenesis (Zoller and Smith, (1983) Methods Enzymol 100:468-500;Zoller and Smith, (1987) Methods Enzymol 154:329-350 (1987); Zoller andSmith, (1982) Nucleic Acids Res 10:6487-6500), phosphorothioate-modifiedDNA mutagenesis (Taylor, et al., (1985) Nucl Acids Res 13:8749-8764;Taylor, et al., (1985) Nucl Acids Res 13:8765-8787 (1985); Nakamaye andEckstein, (1986) Nucl Acids Res 14:9679-9698; Sayers, et al., (1988)Nucl Acids Res 16:791-802 and Sayers, et al., (1988) Nucl Acids Res16:803-814); mutagenesis using gapped duplex DNA (Kramer, et al., (1984)Nucl Acids Res 12:9441-9456; Kramer and Fritz, (1987) Methods Enzymol154:350-367; Kramer, et al., (1988) Nucl Acids Res 16:7207 and Fritz, etal., (1988) Nucl Acids Res 16:6987-6999).

Additional suitable methods include point mismatch repair (Kramer, etal., (1984) Cell 38:879-887), mutagenesis using repair-deficient hoststrains (Carter, et al., (1985) Nucl Acids Res 13:4431-4443 and Carter,(1987) Methods in Enzymol 154:382-403), deletion mutagenesis(Eghtedarzadeh and Henikoff, (1986) Nucl Acids Res 14:5115),restriction-selection and restriction-purification (Wells, et al.,(1986) Phil Trans R Soc Lond A 317:415-423), mutagenesis by total genesynthesis (Nambiar, et al., (1984) Science 223:1299-1301; Sakamar andKhorana, (1988) Nucl Acids Res 14:6361-6372; Wells, et al., (1985) Gene34:315-323 and Grundstrom, et al., (1985) Nucl Acids Res 13:3305-3316),double-strand break repair (Mandecki, (1986) PNAS USA, 83:7177-7181 andArnold, (1993) Curr Opin Biotech 4:450-455). Additional details on manyof the above methods can be found in Methods Enzymol Volume 154, whichalso describes useful controls for trouble-shooting problems withvarious mutagenesis methods.

Development of Oligonucleotide Probes. A further method for identifyingthe toxins and genes of the subject invention is through the use ofoligonucleotide probes. These probes are detectable nucleotidesequences. These sequences may be rendered detectable by virtue of anappropriate radioactive label or may be made inherently fluorescent asdescribed in, for example, U.S. Pat. No. 6,268,132. As is well known inthe art, if the probe molecule and nucleic acid sample hybridize byforming strong base-pairing bonds between the two molecules, it can bereasonably assumed that the probe and sample have substantial sequencehomology. Preferably, hybridization is conducted under stringentconditions by techniques well-known in the art, as described, forexample, in Keller and Manak (1993). Detection of the probe provides ameans for determining in a known manner whether hybridization hasoccurred. Such a probe analysis provides a rapid method for identifyingtoxin-encoding genes of the subject invention. The nucleotide segmentswhich are used as probes according to the invention can be synthesizedusing a DNA synthesizer and standard procedures. These nucleotidesequences can also be used as PCR primers to amplify genes of thesubject invention.

Nucleic acid hybridization. As is well known to those skilled inmolecular biology, similarity of two nucleic acids can be characterizedby their tendency to hybridize. As used herein the terms “stringentconditions” or “stringent hybridization conditions” are intended torefer to conditions under which a probe will hybridize (anneal) to itstarget sequence to a detectably greater degree than to other sequences(e.g. at least 2-fold over background). Stringent conditions aresequence-dependent and will be different in different circumstances. Bycontrolling the stringency of the hybridization and/or washingconditions, target sequences that are 100% complementary to the probecan be identified (homologous probing). Alternatively, stringencyconditions can be adjusted to allow some mismatching in sequences sothat lower degrees of similarity are detected (heterologous probing).Generally, a probe is less than about 1000 nucleotides in length,preferably less than 500 nucleotides in length.

Typically, stringent conditions will be those in which the saltconcentration is less than about 1.5 M Na ion, typically about 0.01 to1.0 M Na ion concentration (or other salts) at pH 7.0 to pH 8.3 and thetemperature is at least about 30° C. for short probes (e.g. 10 to 50nucleotides) and at least about 60° C. for long probes (e.g. greaterthan 50 nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. Exemplary lowstringency conditions include hybridization with a buffer solution of30% to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulfate) at 37°C. and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate)at 50° C. to 55° C. Exemplary moderate stringency conditions includehybridization in 40% to 45% formamide, 1.0 M NaCl, 1% SDS at 37° C. anda wash in 0.5× to 1×SSC at 55° C. to 60° C. Exemplary high stringencyconditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at37° C. and a wash in 0.1×SSC at 60° C. to 65° C. Optionally, washbuffers may comprise about 0.1% to about 1% SDS. Duration ofhybridization is generally less than about 24 hours, usually about 4 to12 hours.

Specificity is typically the function of post-hybridization washes, thecritical factors being the ionic strength and temperature of the finalwash solution. For DNA/DNA hybrids, the thermal melting point (T_(m)) isthe temperature (under defined ionic strength and pH) at which 50% of acomplementary target sequence hybridizes to a perfectly matched probe.T_(m) is reduced by about 1° C. for each 1% of mismatching; thus, T_(m),hybridization conditions, and/or wash conditions can be adjusted tofacilitate annealing of sequences of the desired identity. For example,if sequences with >90% identity are sought, the T_(m) can be decreased10° C. Generally, stringent conditions are selected to be about 5° C.lower than the T_(m) for the specific sequence and its complement at adefined ionic strength and pH. However, highly stringent conditions canutilize a hybridization and/or wash at 1° C., 2° C., 3° C., or 4° C.lower than the T_(m); moderately stringent conditions can utilize ahybridization and/or wash at 6° C., 7° C., 8° C., 9° C., or 10° C. lowerthan the T_(m), and low stringency conditions can utilize ahybridization and/or wash at 11° C., 12° C., 13° C., 14° C., 15° C., or20° C. lower than the T_(m).

T_(m) (in ° C.) may be experimentally determined or may be approximatedby calculation. For DNA-DNA hybrids, the T_(m) can be approximated fromthe equation of Meinkoth and Wahl (1984): T_(m)(° C.)=81.5° C.+16.6(logM)+0.41(% GC)−0.61(% formamide)−500/L; where M is the molarity ofmonovalent cations, % GC is the percentage of guanosine and cytosinenucleotides in the DNA, % formamide is the percentage of formamide inthe hybridization solution, and L is the length of the hybrid in basepairs. Alternatively, the T_(m) is described by the following formula(Beltz et al., 1983): T_(m)(° C.)=81.5° C.+16.6(log[Na+])+0.41(%GC)−0.61(% formamide)−600/L where [Na+] is the molarity of sodium ions,% GC is the percentage of guanosine and cytosine nucleotides in the DNA,% formamide is the percentage of formamide in the hybridizationsolution, and L is the length of the hybrid in base pairs. Using theequations, hybridization and wash compositions, and desired T_(m), thoseof ordinary skill will understand that variations in the stringency ofhybridization and/or wash solutions are inherently described. If thedesired degree of mismatching results in a T_(m) of less than 45° C.(aqueous solution) or 32° C. (formamide solution), it is preferred toincrease the SSC concentration so that a higher temperature can be used.An extensive guide to the hybridization of nucleic acids is found inHybridization with Nucleic Acid Probes Vol. 1 by P. Tijssen (1993,ISBN-10: 0444898840, ISBN-13: 9780444898845 Hardcover) and Ausubel etal. (1995). Also see Sambrook et al. (1989).

Anti-toxin antibodies. Antibodies to the toxins disclosed herein, orfragments of these toxins, can be prepared using standard procedureswell known in the art. Such antibodies are useful to detect the presenceof IRDIG35563 toxins in plant tissues and a variety of other substances.Such antibodies and anti-sera are useful in various methods of detectingthe claimed IRDIG35563 toxins of the invention, and variants orfragments thereof. It is well known that antibodies labeled with areporting group can be used to identify the presence of antigens in avariety of milieus. Antibodies labeled with radioisotopes have been usedin radioimmunoassays to identify, with great precision and sensitivity,the presence of antigens in a variety of biological fluids. Morerecently, enzyme labeled antibodies have been used as a substitute forradiolabeled antibodies in the ELISA assay. Further, antibodiesimmunoreactive to the Bt insecticidal toxin of the present invention canbe bound to an immobilizing substance such as a polystyrene well orparticle and used in immunoassays to determine whether the Bt toxin ispresent in a test sample. Anti-IRDIG35563 antibodies are also used forisolating quantities of IRDIG35563 toxins from recombinant productionsystems or natural sources.

A kit for detecting the presence of an IRDIG35563 polypeptide ordetecting the presence of a nucleotide sequence encoding an IRDIG35563polypeptide in a sample is provided. In one embodiment, the kit providesantibody-based reagents for detecting the presence of an IRDIG35563polypeptide in a tissue sample. In another embodiment, the kit provideslabeled nucleic acid probes useful for detecting the presence of one ormore polynucleotides encoding an IRDIG35563 polypeptide. The kit isprovided along with appropriate reagents and controls for carrying out adetection method, as well as instructions for use of the kit.

Transgenic Expression of IRDIG35563. The subject protein toxins can be“applied” or provided to contact the target insects in a variety ofways. For example, IRDIG35563 toxins can be used as plant-incorporatedprotectants in transgenic plants (produced by and present in the plant).Expression of the toxin genes can also achieve selectivity in specifictissues of the plants, such as the roots, leaves, etc. This can beaccomplished via the use of tissue-specific promoters.

A preferred embodiment of the subject invention is the transformation ofplants with genes encoding the subject insecticidal protein or itsvariants. The transformed plants are resistant to prolonged attack by aninsect target pest by virtue of the presence of controlling amounts ofthe subject insecticidal protein or its variants in the cells of thetransformed plant. By incorporating and expressing genetic material thatencodes an IRDIG35563 toxin into the genome of a plant eaten by aparticular insect pest, the adult or larvae will die after consuming thefood plant. Numerous members of the monocotyledonous and dicotyledonousclassifications have been transformed. Transgenic agronomic crops aswell as fruits and vegetables are of commercial interest. Such cropsinclude, but are not limited to, maize, rice, soybeans, canola,sunflower, alfalfa, sorghum, wheat, cotton, peanuts, tomatoes, potatoes,and the like. Numerous well known techniques exist for introducingforeign genetic material into monocot or dicot plant cells, and forobtaining fertile plants that stably maintain and express the introducedgene.

In one preferred embodiment, IRDIG35563 or a variant is delivered orallythrough a transgenic plant comprising a nucleic acid sequence thatexpresses a toxin of the present invention. The present inventionprovides a method of producing an insect-resistant transgenic plant,comprising introducing a nucleic acid molecule of the invention into theplant wherein the toxin is expressible in the transgenic plant in aneffective amount to control an insect. In a non-limiting example, abasic cloning strategy may be to subclone full length or modifiedIRDIG35563 coding sequences into a plant expression plasmid at Ncol andSacI restriction sites. The resulting plant expression cassettescontaining the appropriate IRDIG35563 coding region under the control ofplant expression elements, (e.g., plant expressible promoters, 3′terminal transcription termination and polyadenylate additiondeterminants, and the like) are subcloned into a binary vector plasmid,utilizing, for example, Gateway® technology or standard restrictionenzyme fragment cloning procedures. LR Clonase™ (Invitrogen, Carlsbad,Calif.) for example, may be used to recombine the full length andmodified gene plant expression cassettes into a binary planttransformation plasmid if the Gateway® technology is utilized. It isconvenient to employ a binary plant transformation vector that harbors abacterial gene that confers resistance to the antibiotic spectinomycinwhen the plasmid is present in E. coli and Agrobacterium cells. It isalso convenient to employ a binary vector plasmid that contains aplant-expressible selectable marker gene that is functional in thedesired host plants. Examples of plant-expressible selectable markergenes include but are not limited to those that encode theaminoglycoside phosphotransferase gene (aphII) of transposon Tn5, whichconfers resistance to the antibiotics kanamycin, neomycin and G418, aswell as those genes which code for resistance or tolerance toglyphosate; hygromycin; methotrexate; phosphinothricin (bialaphos),imidazolinones, sulfonylureas and triazolopyrimidine herbicides, such aschlorosulfuron, bromoxynil, dalapon and the like.

Alternatively, the plasmid structure of the binary plant transformationvector containing the IRDIG35563 gene insert is performed by restrictiondigest fingerprint mapping of plasmid DNA prepared from candidateAgrobacterium isolates by standard molecular biology methods well knownto those skilled in the art of Agrobacterium manipulation.

The use of the term “nucleotide constructs” herein is not intended tolimit the embodiments to nucleotide constructs comprising DNA.Nucleotide constructs particularly polynucleotides and oligonucleotidescomposed of ribonucleotides and combinations of ribonucleotides anddeoxyribonucleotides may also be employed in the methods disclosedherein. The nucleotide constructs, nucleic acids, and nucleotidesequences of the embodiments additionally encompass all complementaryforms of such constructs, molecules, and sequences. Further, thenucleotide constructs, nucleotide molecules, and nucleotide sequences ofthe embodiments encompass all nucleotide constructs, molecules, andsequences which can be employed in the methods of the embodiments fortransforming plants including, but not limited to, those comprised ofdeoxyribonucleotides, ribonucleotides, and combinations thereof. Suchdeoxyribonucleotides and ribonucleotides include both naturallyoccurring molecules and synthetic analogues. The nucleotide constructs,nucleic acids, and nucleotide sequences of the embodiments alsoencompass all forms of nucleotide constructs including, but not limitedto, single-stranded forms, double-stranded forms, hairpins,stem-and-loop structures and the like.

A further embodiment relates to a transformed organism such as anorganism selected from plant and insect cells, bacteria, yeast,baculovirus, protozoa, nematodes and algae. The transformed organismcomprises a DNA molecule of the embodiments, an expression cassettecomprising the DNA molecule or a vector comprising the expressioncassette, which may be stably incorporated into the genome of thetransformed organism.

The sequences of the embodiments are provided in DNA constructs forexpression in the organism of interest. The construct will include 5′and 3′ regulatory sequences operably linked to a sequence of theembodiments. The term “operably linked” as used herein refers to afunctional linkage between a promoter and a second sequence, wherein thepromoter sequence initiates and mediates transcription of the DNAsequence corresponding to the second sequence. Generally, operablylinked means that the nucleic acid sequences being linked are contiguousand where necessary to join two protein coding regions in the samereading frame. The construct may additionally contain at least oneadditional gene to be cotransformed into the organism. Alternatively,the additional gene(s) can be provided on multiple DNA constructs.

Such a DNA construct is provided with a plurality of restriction sitesfor insertion of the IRDIG35563 polypeptide gene sequence of thedisclosure to be under the transcriptional regulation of the regulatoryregions. The DNA construct may additionally contain selectable markergenes.

The DNA construct will generally include in the 5′ to 3′ direction oftranscription: a transcriptional and translational initiation region(i.e., a promoter), a DNA sequence of the embodiments, and atranscriptional and translational termination region (i.e., terminationregion) functional in the organism serving as a host. Thetranscriptional initiation region (i.e., the promoter) may be native,analogous, foreign or heterologous to the host organism and/or to thesequence of the embodiments. Additionally, the promoter may be thenatural sequence or alternatively a synthetic sequence. The term“foreign” as used herein indicates that the promoter is not found in thenative organism into which the promoter is introduced. Where thepromoter is “foreign” or “heterologous” to the sequence of theembodiments, it is intended that the promoter is not the native ornaturally occurring promoter for the operably linked sequence of theembodiments. As used herein, a chimeric gene comprises a coding sequenceoperably linked to a transcription initiation region that isheterologous to the coding sequence. Where the promoter is a native ornatural sequence, the expression of the operably linked sequence isaltered from the wild-type expression, which results in an alteration inphenotype.

In some embodiments, the DNA construct comprises a polynucleotideencoding an IRDIG35563 polypeptide of the embodiments.

In some embodiments, the DNA construct comprises a polynucleotideencoding a fusion protein comprising an IRDIG35563 polypeptide of theembodiments.

In some embodiments, the DNA construct may also include atranscriptional enhancer sequence. As used herein, the term an“enhancer” refers to a DNA sequence which can stimulate promoteractivity, and may be an innate element of the promoter or a heterologouselement inserted to enhance the level or tissue-specificity of apromoter. Various enhancers including, introns with gene expressionenhancing properties in plants (US Patent Application Publication Number2009/0144863, the ubiquitin intron (i.e., the maize ubiquitin intron 1(see, for example, NCBI sequence S94464)), the omega enhancer or theomega prime enhancer (Gallie, et al., (1989) Molecular Biology of RNAed. Cech (Liss, New York) 237-256 and Gallie, et al., (1987) Gene60:217-25), the CaMV 35S enhancer (see, e.g., Benfey, et al., (1990)EMBO J. 9:1685-96) and the enhancers of U.S. Pat. No. 7,803,992 may alsobe used, each of which is incorporated by reference. The above list oftranscriptional enhancers is not meant to be limiting. Any appropriatetranscriptional enhancer can be used in the embodiments.

The termination region may be native with the transcriptional initiationregion, may be native with the operably linked DNA sequence of interest,may be native with the plant host or may be derived from another source(i.e., foreign or heterologous to the promoter, the sequence ofinterest, the plant host or any combination thereof).

Convenient termination regions are available from the Ti-plasmid of A.tumefaciens, such as the octopine synthase and nopaline synthasetermination regions. See also, Guerineau, et al., (1991) Mol. Gen.Genet. 262:141-144; Proudfoot, (1991) Cell 64:671-674; Sanfacon, et al.,(1991) Genes Dev. 5:141-149; Mogen, et al., (1990) Plant Cell2:1261-1272; Munroe, et al., (1990) Gene 91:151-158; Ballas, et al.,(1989) Nucleic Acids Res. 17:7891-7903 and Joshi, et al., (1987) NucleicAcid Res. 15:9627-9639.

Where appropriate, a nucleic acid may be optimized for increasedexpression in the host organism. Thus, where the host organism is aplant, the synthetic nucleic acids can be synthesized usingplant-preferred codons for improved expression. See, for example,Campbell and Gowri, (1990) Plant Physiol. 92:1-11 for a discussion ofhost-preferred usage. For example, although nucleic acid sequences ofthe embodiments may be expressed in both monocotyledonous anddicotyledonous plant species, sequences can be modified to account forthe specific preferences and GC content preferences of monocotyledons ordicotyledons as these preferences have been shown to differ (Murray etal. (1989) Nucleic Acids Res. 17:477-498). Thus, the maize-preferredcodon for a particular amino acid may be derived from known genesequences from maize. Maize usage for 28 genes from maize plants islisted in Table 4 of Murray, et al., supra. Methods for synthesizingplant-preferred genescan be found in Murray, et al., (1989) NucleicAcids Res. 17:477-498, and Liu H et al. Mo. Bio Rep 37:677-684, 2010,herein incorporated by reference. A Zea maize usage table can be alsofound at kazusa.or.jp//cgi-bin/show.cgi?species=4577, which can beaccessed using the www prefix.

A Glycine max usage table can be found atkazusa.or.jp//cgi-bin/show.cgi?species=3847&aa=1&style=N, which can beaccessed using the www prefix.

In some embodiments, the recombinant nucleic acid molecule encoding anIRDIG35563 polypeptide has maize optimized codons. In some embodiments,the recombinant nucleic acid molecule encoding an IRDIG35563 polypeptidehas soy optimized codons.

Additional sequence modifications are known to enhance gene expressionin a cellular host. These include elimination of sequences encodingspurious polyadenylation signals, exon-intron splice site signals,transposon-like repeats, and other well-characterized sequences that maybe deleterious to gene expression. The GC content of the sequence may beadjusted to levels average for a given cellular host, as calculated byreference to known genes expressed in the host cell. The term “hostcell” as used herein refers to a cell which contains a vector andsupports the replication and/or expression of the expression vector isintended. Host cells may be prokaryotic cells such as E. coli oreukaryotic cells such as yeast, insect, amphibian or mammalian cells ormonocotyledonous or dicotyledonous plant cells. An example of amonocotyledonous host cell is a maize host cell. When possible, thesequence is modified to avoid predicted hairpin secondary mRNAstructures.

The expression cassettes may additionally contain 5′ leader sequences.Such leader sequences can act to enhance translation. Translationleaders include: picornavirus leaders, for example, EMCV leader(Encephalomyocarditis 5′ noncoding region) (Elroy-Stein, et al., (1989)Proc. Natl. Acad. Sci. USA 86:6126-6130); potyvirus leaders, forexample, TEV leader (Tobacco Etch Virus) (Gallie, et al., (1995) Gene165(2):233-238), MDMV leader (Maize Dwarf Mosaic Virus), humanimmunoglobulin heavy-chain binding protein (BiP) (Macejak, et al.,(1991) Nature 353:90-94); untranslated leader from the coat protein mRNAof alfalfa mosaic virus (AMV RNA 4) (Jobling, et al., (1987) Nature325:622-625); tobacco mosaic virus leader (TMV) (Gallie, et al., (1989)in Molecular Biology of RNA, ed. Cech (Liss, New York), pp. 237-256) andmaize chlorotic mottle virus leader (MCMV) (Lommel, et al., (1991)Virology 81:382-385). See also, Della-Cioppa, et al., (1987) PlantPhysiol. 84:965-968. Such constructs may also contain a “signalsequence” or “leader sequence” to facilitate co-translational orpost-translational transport of the peptide to certain intracellularstructures such as the chloroplast (or other plastid), endoplasmicreticulum or Golgi apparatus.

“Signal sequence” as used herein refers to a sequence that is known orsuspected to result in cotranslational or post-translational peptidetransport across the cell membrane. In eukaryotes, this typicallyinvolves secretion into the Golgi apparatus, with some resultingglycosylation. Insecticidal toxins of bacteria are often synthesized asprotoxins, which are proteolytically activated in the gut of the targetpest (Chang, (1987) Methods Enzymol. 153:507-516). In some embodiments,the signal sequence is in the native sequence or may be derived from asequence of the embodiments. “Leader sequence” as used herein refers toany sequence that when translated, results in an amino acid sequencesufficient to trigger co-translational transport of the peptide chain toa subcellular organelle. Thus, this includes leader sequences targetingtransport and/or glycosylation by passage into the endoplasmicreticulum, passage to vacuoles, plastids including chloroplasts,mitochondria, and the like. Nuclear-encoded proteins targeted to thechloroplast thylakoid lumen compartment have a characteristic bipartitetransit peptide, composed of a stromal targeting signal peptide and alumen targeting signal peptide. The stromal targeting information is inthe amino-proximal portion of the transit peptide. The lumen targetingsignal peptide is in the carboxyl-proximal portion of the transitpeptide, and contains all the information for targeting to the lumen.Recent research in proteomics of the higher plant chloroplast hasachieved in the identification of numerous nuclear-encoded lumenproteins (Kieselbach et al. FEBS LETT 480:271-276, 2000; Peltier et al.Plant Cell 12:319-341, 2000; Bricker et al. Biochim. Biophys Acta1503:350-356, 2001), the lumen targeting signal peptide of which canpotentially be used in accordance with the present disclosure. About 80proteins from Arabidopsis, as well as homologous proteins from spinachand garden pea, are reported by Kieselbach et al., PhotosynthesisResearch, 78:249-264, 2003. Table 2 of this publication, which isincorporated into the description herewith by reference, discloses 85proteins from the chloroplast lumen, identified by their accessionnumber (see also US Patent Application Publication 2009/09044298). Inaddition, the recently published draft version of the rice genome (Goffet al, Science 296:92-100, 2002) is a suitable source for lumentargeting signal peptide which may be used in accordance with thepresent disclosure.

Suitable chloroplast transit peptides (CTP) include chimeric CT'scomprising but not limited to, an N-terminal domain, a central domain ora C-terminal domain from a CTP from Oryza sativa 1-decoy-Dxylose-5-Phosphate Synthase Oryza sativa-Superoxide dismutase Oryzasativa-soluble starch synthase Oryza sativa-NADP-dependent Malic acidenzyme Oryza sativa-Phospho-2-dehydro-3-deoxyheptonate Aldolase 2 Oryzasativa-L-Ascorbate peroxidase 5 Oryza sativa-Phosphoglucan waterdikinase, Zea Mays ssRUBISCO, Zea Mays-beta-glucosidase, Zea Mays-Malatedehydrogenase, Zea Mays Thioredoxin M-type US Patent ApplicationPublication 2012/0304336).

The IRDIG35563 polypeptide gene to be targeted to the chloroplast may beoptimized for expression in the chloroplast to account for differencesin usage between the plant nucleus and this organelle. In this manner,the nucleic acids of interest may be synthesized usingchloroplast-preferred sequences.

In preparing the expression cassette, the various DNA fragments may bemanipulated to provide for the DNA sequences in the proper orientationand, as appropriate, in the proper reading frame. Toward this end,adapters or linkers may be employed to join the DNA fragments or othermanipulations may be involved to provide for convenient restrictionsites, removal of superfluous DNA, removal of restriction sites or thelike. For this purpose, in vitro mutagenesis, primer repair,restriction, annealing, resubstitutions, e.g., transitions andtransversions, may be involved.

Several promoters can be used in the practice of the embodiments. Thepromoters can be selected based on the desired outcome. The nucleicacids can be combined with constitutive, tissue-preferred, inducible orother promoters for expression in the host organism. Suitableconstitutive promoters for use in a plant host cell include, forexample, the core promoter of the Rsyn7 promoter and other constitutivepromoters disclosed in WO 1999/43838 and U.S. Pat. No. 6,072,050; thecore CaMV 35S promoter (Odell, et al., (1985) Nature 313:810-812); riceactin (McElroy, et al., (1990) Plant Cell 2:163-171); ubiquitin(Christensen, et al., (1989) Plant Mol. Biol. 12:619-632 andChristensen, et al., (1992) Plant Mol. Biol. 18:675-689); pEMU (Last, etal., (1991) Theor. Appl. Genet. 81:581-588); MAS (Velten, et al., (1984)EMBO J. 3:2723-2730); ALS promoter (U.S. Pat. No. 5,659,026) and thelike. Other constitutive promoters include, for example, those discussedin U.S. Pat. Nos. 5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785;5,399,680; 5,268,463; 5,608,142 and 6,177,611.

Depending on the desired outcome, it may be beneficial to express thegene from an inducible promoter. Of particular interest for regulatingthe expression of the nucleotide sequences of the embodiments in plantsare wound-inducible promoters. Such wound-inducible promoters, mayrespond to damage caused by insect feeding, and include potatoproteinase inhibitor (pin II) gene (Ryan, (1990) Ann. Rev. Phytopath.28:425-449; Duan, et al., (1996) Nature Biotechnology 14:494-498); wun1and wun2, U.S. Pat. No. 5,428,148; win1 and win2 (Stanford, et al.,(1989)Mol. Gen. Genet. 215:200-208); systemin (McGurl, et al., (1992)Science 225:1570-1573); WIP1 (Rohmeier, et al., (1993) Plant Mol. Biol.22:783-792; Eckelkamp, et al., (1993) FEBS Letters 323:73-76); MPI gene(Corderok, et al., (1994) Plant J. 6(2):141-150) and the like, hereinincorporated by reference.

Additionally, pathogen-inducible promoters may be employed in themethods and nucleotide constructs of the embodiments. Suchpathogen-inducible promoters include those from pathogenesis-relatedproteins (PR proteins), which are induced following infection by apathogen; e.g., PR proteins, SAR proteins, beta-1,3-glucanase,chitinase, etc. See, for example, Redolfi, et al., (1983) Neth. J. PlantPathol. 89:245-254; Uknes, et al., (1992) Plant Cell 4: 645-656 and VanLoon, (1985) Plant Mol. Virol. 4:111-116. See also, WO 1999/43819,herein incorporated by reference.

Of interest are promoters that are expressed locally at or near the siteof pathogen infection. See, for example, Marineau, et al., (1987) PlantMol. Biol. 9:335-342; Matton, et al., (1989) Molecular Plant-MicrobeInteractions 2:325-331; Somsisch, et al., (1986) Proc. Natl. Acad. Sci.USA 83:2427-2430; Somsisch, et al., (1988)Mol. Gen. Genet. 2:93-98 andYang, (1996) Proc. Natl. Acad. Sci. USA 93:14972-14977. See also, Chen,et al., (1996) Plant J. 10:955-966; Zhang, et al., (1994) Proc. Natl.Acad. Sci. USA 91:2507-2511; Warner, et al., (1993) Plant J. 3:191-201;Siebertz, et al., (1989) Plant Cell 1:961-968; U.S. Pat. No. 5,750,386(nematode-inducible) and the references cited therein. Of particularinterest is the inducible promoter for the maize PRms gene, whoseexpression is induced by the pathogen Fusarium moniliforme (see, forexample, Cordero, et al., (1992) Physiol. Mol. Plant Path. 41:189-200).

Chemical-regulated promoters can be used to modulate the expression of agene in a plant through the application of an exogenous chemicalregulator. Depending upon the objective, the promoter may be achemical-inducible promoter, where application of the chemical inducesgene expression or a chemical-repressible promoter, where application ofthe chemical represses gene expression. Chemical-inducible promotersinclude but are not limited to, the maize In2-2 promoter, which isactivated by benzenesulfonamide herbicide safeners, the maize GSTpromoter, which is activated by hydrophobic electrophilic compounds thatare used as pre-emergent herbicides, and the tobacco PR-la promoter,which is activated by salicylic acid. Other chemical-regulated promotersof interest include steroid-responsive promoters (see, for example, theglucocorticoid-inducible promoter in Schena, et al., (1991) Proc. Natl.Acad. Sci. USA 88:10421-10425 and McNellis, et al., (1998) Plant J.14(2):247-257) and tetracycline-inducible and tetracycline-repressiblepromoters (see, for example, Gatz, et al., (1991) Mol. Gen. Genet.227:229-237 and U.S. Pat. Nos. 5,814,618 and 5,789,156), hereinincorporated by reference.

Tissue-preferred promoters can be utilized to target IRDIG35563polypeptide expression within a particular plant tissue.Tissue-preferred promoters include those discussed in Yamamoto, et al.,(1997) Plant J. 12(2)255-265; Kawamata, et al., (1997) Plant CellPhysiol. 38(7):792-803; Hansen, et al., (1997) Mol. Gen Genet.254(3):337-343; Russell, et al., (1997) Transgenic Res. 6(2):157-168;Rinehart, et al., (1996) Plant Physiol. 112(3):1331-1341; Van Camp, etal., (1996) Plant Physiol. 112(2):525-535; Canevascini, et al., (1996)Plant Physiol. 112(2):513-524; Yamamoto, et al., (1994) Plant CellPhysiol. 35(5):773-778; Lam, (1994) Results Probl. Cell Differ.20:181-196; Orozco, et al., (1993) Plant Mol Biol. 23(6):1129-1138;Matsuoka, et al., (1993) Proc Natl. Acad. Sci. USA 90(20):9586-9590 andGuevara-Garcia, et al., (1993) Plant J. 4(3):495-505. Such promoters canbe modified, if necessary, for weak expression.

Leaf-preferred promoters can be found in Yamamoto, et al., (1997) PlantJ. 12(2):255-265; Kwon, et al., (1994) Plant Physiol. 105:357-67;Yamamoto, et al., (1994) Plant Cell Physiol. 35(5):773-778; Gotor, etal., (1993) Plant J. 3:509-18; Orozco, et al., (1993) Plant Mol. Biol.23(6):1129-1138 and Matsuoka, et al., (1993) Proc. Natl. Acad. Sci. USA90(20):9586-9590.

Root-preferred or root-specific promoters are known and can be selectedfrom the many available from the literature or isolated de novo fromvarious compatible species. See, for example, Hire, et al., (1992) PlantMol. Biol. 20(2):207-218 (soybean root-specific glutamine synthetasegene); Keller and Baumgartner, (1991) Plant Cell 3(10):1051-1061(root-specific control element in the GRP 1.8 gene of French bean);Sanger, et al., (1990) Plant Mol. Biol. 14(3):433-443 (root-specificpromoter of the mannopine synthase (MAS) gene of Agrobacteriumtumefaciens) and Miao, et al., (1991) Plant Cell 3(1):11-22 (full-lengthcDNA clone encoding cytosolic glutamine synthetase (GS), which isexpressed in roots and root nodules of soybean). See also, Bogusz, etal., (1990) Plant Cell 2(7):633-641, where two root-specific promotersisolated from hemoglobin genes from the nitrogen-fixing nonlegumeParasponia andersonii and the related non-nitrogen-fixing nonlegumeTrema tomentosa are described. The promoters of these genes were linkedto a β-glucuronidase reporter gene and introduced into both thenonlegume Nicotiana tabacum and the legume Lotus corniculatus, and inboth instances root-specific promoter activity was preserved. Leach andAoyagi, (1991) describe their analysis of the promoters of the highlyexpressed rolC and rolD root-inducing genes of Agrobacterium rhizogenes(see, Plant Science (Limerick) 79(1):69-76). They concluded thatenhancer and tissue-preferred DNA determinants are dissociated in thosepromoters. Teeri, et al., (1989) used gene fusion to lacZ to show thatthe Agrobacterium T-DNA gene encoding octopine synthase is especiallyactive in the epidermis of the root tip and that the TR2′ gene is rootspecific in the intact plant and stimulated by wounding in leaf tissue,an especially desirable combination of characteristics for use with aninsecticidal or larvicidal gene (see, EMBO J 8(2):343-350). The TR1′gene fused to nptII (neomycin phosphotransferase II) showed similarcharacteristics. Additional root-preferred promoters include theVfENOD-GRP3 gene promoter (Kuster, et al., (1995) Plant Mol. Biol.29(4):759-772) and rolB promoter (Capana, et al., (1994) Plant Mol.Biol. 25(4):681-691. See also, U.S. Pat. Nos. 5,837,876; 5,750,386;5,633,363; 5,459,252; 5,401,836; 5,110,732 and 5,023,179. Arabidopsisthaliana root-preferred regulatory sequences are disclosed inUS20130117883.

“Seed-preferred” promoters include both “seed-specific” promoters (thosepromoters active during seed development such as promoters of seedstorage proteins) as well as “seed-germinating” promoters (thosepromoters active during seed germination). See, Thompson, et al., (1989)BioEssays 10:108, herein incorporated by reference. Such seed-preferredpromoters include, but are not limited to, Cim1 (cytokinin-inducedmessage); cZ19B1 (maize 19 kDa zein); and milps(myo-inositol-1-phosphate synthase) (see, U.S. Pat. No. 6,225,529,herein incorporated by reference). Gamma-zein and Glb-1 areendosperm-specific promoters. For dicots, seed-specific promotersinclude, but are not limited to, Kunitz trypsin inhibitor 3 (KTi3)(Jofuku and Goldberg, (1989) Plant Cell 1:1079-1093), bean β-phaseolin,napin, β-conglycinin, glycinin 1, soybean lectin, cruciferin, and thelike. For monocots, seed-specific promoters include, but are not limitedto, maize 15 kDa zein, 22 kDa zein, 27 kDa zein, g-zein, waxy, shrunken1, shrunken 2, globulin 1, etc. See also, WO 2000/12733, whereseed-preferred promoters from end1 and end2 genes are disclosed; hereinincorporated by reference. In dicots, seed specific promoters includebut are not limited to seed coat promoter from Arabidopsis, pBAN; andthe early seed promoters from Arabidopsis, p26, p63, and p63tr (U.S.Pat. Nos. 7,294,760 and 7,847,153). A promoter that has “preferred”expression in a particular tissue is expressed in that tissue to agreater degree than in at least one other plant tissue. Sometissue-preferred promoters show expression almost exclusively in theparticular tissue.

Where low level expression is desired, weak promoters will be used.Generally, the term “weak promoter” as used herein refers to a promoterthat drives expression of a coding sequence at a low level. By low levelexpression at levels of between about 1/1000 transcripts to about1/100,000 transcripts to about 1/500,000 transcripts is intended.Alternatively, it is recognized that the term “weak promoters” alsoencompasses promoters that drive expression in only a few cells and notin others to give a total low level of expression. Where a promoterdrives expression at unacceptably high levels, portions of the promotersequence can be deleted or modified to decrease expression levels.

Such weak constitutive promoters include, for example the core promoterof the Rsyn7 promoter (WO 1999/43838 and U.S. Pat. No. 6,072,050), thecore 35S CaMV promoter, and the like. Other constitutive promotersinclude, for example, those disclosed in U.S. Pat. Nos. 5,608,149;5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680; 5,268,463;5,608,142 and 6,177,611, herein incorporated by reference.

The above list of promoters is not meant to be limiting. Any appropriatepromoter can be used in the embodiments.

Generally, the expression cassette will comprise a selectable markergene for the selection of transformed cells. Selectable marker genes areutilized for the selection of transformed cells or tissues. Marker genesinclude genes encoding antibiotic resistance, such as those encodingneomycin phosphotransferase II (NEO) and hygromycin phosphotransferase(HPT), as well as genes conferring resistance to herbicidal compounds,such as glufosinate ammonium, bromoxynil, imidazolinones and2,4-dichlorophenoxyacetate (2,4-D). Additional examples of suitableselectable marker genes include, but are not limited to, genes encodingresistance to chloramphenicol (Herrera Estrella, et al., (1983) EMBO J.2:987-992); methotrexate (Herrera Estrella, et al., (1983) Nature303:209-213 and Meijer, et al., (1991) Plant Mol. Biol. 16:807-820);streptomycin (Jones, et al., (1987)Mol. Gen. Genet. 210:86-91);spectinomycin (Bretagne-Sagnard, et al., (1996) Transgenic Res.5:131-137); bleomycin (Hille, et al., (1990) Plant Mol. Biol.7:171-176); sulfonamide (Guerineau, et al., (1990) Plant Mol. Biol.15:127-136); bromoxynil (Stalker, et al., (1988) Science 242:419-423);glyphosate (Shaw, et al., (1986) Science 233:478-481 and U.S. patentapplication Ser. Nos. 10/004,357 and 10/427,692); phosphinothricin(DeBlock, et al., (1987) EMBO J. 6:2513-2518). See generally, Yarranton,(1992) Curr. Opin. Biotech. 3:506-511; Christopherson, et al., (1992)Proc. Natl. Acad Sci. USA 89:6314-6318; Yao, et al., (1992) Cell71:63-72; Reznikoff, (1992) Mol. Microbiol. 6:2419-2422; Barkley, etal., (1980) in The Operon, pp. 177-220; Hu, et al., (1987) Cell48:555-566; Brown, et al., (1987) Cell 49:603-612; Figge, et al., (1988)Cell 52:713-722; Deuschle, et al., (1989) Proc. Natl. Acad Sci. USA86:5400-5404; Fuerst, et al., (1989) Proc. Natl. Acad Sci. USA86:2549-2553; Deuschle, et al., (1990) Science 248:480-483; Gossen,(1993) Ph.D. Thesis, University of Heidelberg; Reines, et al., (1993)Proc. Natl. Acad Sci. USA 90:1917-1921; Labow, et al., (1990) Mol. Cell.Biol. 10:3343-3356; Zambretti, et al., (1992)Proc. Natl. Acad Sci. USA89:3952-3956; Baim, et al., (1991) Proc. Natl. Acad Sci. USA88:5072-5076; Wyborski, et al., (1991) Nucleic Acids Res. 19:4647-4653;Hillenand-Wissman, (1989) Topics Mol. Struc. Biol. 10:143-162;Degenkolb, et al., (1991) Antimicrob. Agents Chemother. 35:1591-1595;Kleinschnidt, et al., (1988) Biochemistry 27:1094-1104; Bonin, (1993)Ph.D. Thesis, University of Heidelberg; Gossen, et al., (1992) Proc.Natl. Acad. Sci. USA 89:5547-5551; Oliva, et al., (1992) Antimicrob.Agents Chemother. 36:913-919; Hlavka, et al., (1985) Handbook ofExperimental Pharmacology, Vol. 78 (Springer-Verlag, Berlin) and Gill,et al., (1988) Nature 334:721-724. Such disclosures are hereinincorporated by reference.

The above list of selectable marker genes is not meant to be limiting.Any selectable marker gene can be used in the embodiments.

Those skilled in the art of obtaining transformed plants viaAgrobacterium-mediated transformation methods will understand that otherAgrobacterium strains besides Z707S may be used, and the choice ofstrain may depend upon the identity of the host plant species to betransformed.

The methods of the embodiments involve introducing a polypeptide orpolynucleotide into a plant. “Introducing” as used herein meanspresenting to the plant the polynucleotide or polypeptide in such amanner that the sequence gains access to the interior of a cell of theplant. The methods of the embodiments do not depend on a particularmethod for introducing a polynucleotide or polypeptide into a plant,only that the polynucleotide or polypeptides gains access to theinterior of at least one cell of the plant. Methods for introducingpolynucleotide or polypeptides into plants include but not limited to,stable transformation methods, transient transformation methods, andvirus-mediated methods.

“Stable transformation” is as used herein means that the nucleotideconstruct introduced into a plant integrates into the genome of theplant and is capable of being inherited by the progeny thereof.“Transient transformation” as used herein means that a polynucleotide isintroduced into the plant and does not integrate into the genome of theplant or a polypeptide is introduced into a plant. “Plant” as usedherein refers to whole plants, plant organs (e.g., leaves, stems, roots,etc.), seeds, plant cells, propagules, embryos and progeny of the same.Plant cells can be differentiated or undifferentiated (e.g. callus,suspension culture cells, protoplasts, leaf cells, root cells, phloemcells and pollen).

Transformation protocols as well as protocols for introducing nucleotidesequences into plants may vary depending on the type of plant or plantcell, i.e., monocot or dicot, targeted for transformation. Suitablemethods of introducing nucleotide sequences into plant cells andsubsequent insertion into the plant genome include microinjection(Crossway, et al., (1986) Biotechniques 4:320-334), electroporation(Riggs, et al., (1986) Proc. Natl. Acad. Sci. USA 83:5602-5606),Agrobacterium-mediated transformation (U.S. Pat. Nos. 5,563,055 and5,981,840), direct gene transfer (Paszkowski, et al., (1984) EMBO J.3:2717-2722) and ballistic particle acceleration (see, for example, U.S.Pat. Nos. 4,945,050; 5,879,918; 5,886,244 and 5,932,782; Tomes, et al.,(1995) in Plant Cell, Tissue, and Organ Culture: Fundamental Methods,ed. Gamborg and Phillips, (Springer-Verlag, Berlin) and McCabe, et al.,(1988) Biotechnology 6:923-926) and Led transformation (WO 00/28058).For potato transformation see, Tu, et al., (1998) Plant MolecularBiology 37:829-838 and Chong, et al., (2000) Transgenic Research9:71-78. Additional transformation procedures can be found inWeissinger, et al., (1988) Ann. Rev. Genet. 22:421-477; Sanford, et al.,(1987) Particulate Science and Technology 5:27-37 (onion); Christou, etal., (1988) Plant Physiol. 87:671-674 (soybean); McCabe, et al., (1988)Bio Technology 6:923-926 (soybean); Finer and McMullen, (1991) In VitroCell Dev. Biol. 27P:175-182 (soybean); Singh, et al., (1998) Theor.Appl. Genet. 96:319-324 (soybean); Datta, et al., (1990) Biotechnology8:736-740 (rice); Klein, et al., (1988) Proc. Natl. Acad. Sci. USA85:4305-4309 (maize); Klein, et al., (1988) Biotechnology 6:559-563(maize); U.S. Pat. Nos. 5,240,855; 5,322,783 and 5,324,646; Klein, etal., (1988) Plant Physiol. 91:440-444 (maize); Fromm, et al., (1990)Biotechnology 8:833-839 (maize); Hooykaas-Van Slogteren, et al., (1984)Nature (London) 311:763-764; U.S. Pat. No. 5,736,369 (cereals);Bytebier, et al., (1987) Proc. Natl. Acad. Sci. USA 84:5345-5349(Liliaceae); De Wet, et al., (1985) in The Experimental Manipulation ofOvule Tissues, ed. Chapman, et al., (Longman, New York), pp. 197-209(pollen); Kaeppler, et al., (1990) Plant Cell Reports 9:415-418 andKaeppler, et al., (1992) Theor. Appl. Genet. 84:560-566(whisker-mediated transformation); D'Halluin, et al., (1992) Plant Cell4:1495-1505 (electroporation); Li, et al., (1993) Plant Cell Reports12:250-255 and Christou and Ford, (1995) Annals of Botany 75:407-413(rice); Osjoda, et al., (1996) Nature Biotechnology 14:745-750 (maizevia Agrobacterium tumefaciens); all of which are herein incorporated byreference.

In specific embodiments, the sequences of the embodiments can beprovided to a plant using a variety of transient transformation methods.Such transient transformation methods include, but are not limited to,the introduction of the IRDIG35563 polynucleotide or variants andfragments thereof directly into the plant or the introduction of theIRDIG35563 polypeptide transcript into the plant. Such methods include,for example, microinjection or particle bombardment. See, for example,Crossway, et al., (1986) Mol Gen. Genet. 202:179-185; Nomura, et al.,(1986) Plant Sci. 44:53-58; Hepler, et al., (1994) Proc. Natl. Acad.Sci. 91:2176-2180 and Hush, et al., (1994) The Journal of Cell Science107:775-784, all of which are herein incorporated by reference.Alternatively, the IRDIG35563 polynucleotide can be transientlytransformed into the plant using techniques in viral vector system andthe precipitation of the polynucleotide in a manner that precludessubsequent release of the DNA. Thus, transcription from theparticle-bound DNA can occur, but the frequency with which it isreleased to become integrated into the genome is greatly reduced. Suchmethods include the use of particles coated with polyethylimine (PEI;Sigma #P3143).

Methods for the targeted insertion of a polynucleotide at a specificlocation in the plant genome include the insertion of the polynucleotideat a desired genomic location is achieved using a site-specificrecombination system. See, for example, WO 1999/25821, WO 1999/25854, WO1999/25840, WO 1999/25855 and WO 1999/25853, all of which are hereinincorporated by reference. Briefly, the polynucleotide of theembodiments can be contained in transfer cassette flanked by twonon-identical recombination sites. The transfer cassette is introducedinto a plant have stably incorporated into its genome a target sitewhich is flanked by two non-identical recombination sites thatcorrespond to the sites of the transfer cassette. An appropriaterecombinase is provided and the transfer cassette is integrated at thetarget site. The polynucleotide of interest is thereby integrated at aspecific chromosomal position in the plant genome.

Plant transformation vectors may be comprised of one or more DNA vectorsneeded for achieving plant transformationincluding plant transformationvectors that are comprised of more than one contiguous DNA segment.These vectors are often referred to as “binary vectors”. Binary vectorsas well as vectors with helper plasmids are most often used forAgrobacterium-mediated transformation, where the size and complexity ofDNA segments needed to achieve efficient transformation is quite large,and it is advantageous to separate functions onto separate DNAmolecules. Binary vectors typically contain a plasmid vector thatcontains the cis-acting sequences required for T-DNA transfer (such asleft border and right border), a selectable marker that is engineered tobe capable of expression in a plant cell, and a “gene of interest” (agene engineered to be capable of expression in a plant cell for whichgeneration of transgenic plants is desired). Also present on thisplasmid vector are sequences required for bacterial replication. Thecis-acting sequences are arranged in a fashion to allow efficienttransfer into plant cells and expression therein. For example, theselectable marker gene and the pesticidal gene are located between theleft and right borders. Often a second plasmid vector contains thetrans-acting factors that mediate T-DNA transfer from Agrobacterium toplant cells. This plasmid often contains the virulence functions (Virgenes) that allow infection of plant cells by Agrobacterium, andtransfer of DNA by cleavage at border sequences and vir-mediated DNAtransfer (Hellens and Mullineaux, (2000) Trends in Plant Science5:446-451). Several types of Agrobacterium strains (e.g. LBA4404,GV3101, EHA101, EHA105, etc.) can be used for plant transformation. Thesecond plasmid vector is not necessary for transforming the plants byother methods such as microprojection, microinjection, electroporation,polyethylene glycol, etc.

In general, plant transformation methods involve transferringheterologous DNA into target plant cells (e.g., immature or matureembryos, suspension cultures, undifferentiated callus, protoplasts,etc.), followed by applying a maximum threshold level of appropriateselection (depending on the selectable marker gene) to recover thetransformed plant cells from a group of untransformed cell mass.Following integration of heterologous foreign DNA into plant cells, onethen applies a maximum threshold level of appropriate selection in themedium to kill the untransformed cells and separate and proliferate theputatively transformed cells that survive from this selection treatmentby transferring regularly to a fresh medium. By continuous passage andchallenge with appropriate selection, one can identify and proliferatethe cells that are transformed with the plasmid vector. Molecular andbiochemical methods can then be used to confirm the presence of theintegrated heterologous gene of interest into the genome of thetransgenic plant.

Explants are typically transferred to a fresh supply of the same mediumand cultured routinely. Subsequently, the transformed cells aredifferentiated into shoots after placing on regeneration mediumsupplemented with a maximum threshold level of selecting agent. Theshoots are then transferred to a selective rooting medium for recoveringrooted shoot or plantlet. The transgenic plantlet then grows into amature plant and produces fertile seeds (e.g., Hiei, et al., (1994) ThePlant Journal 6:271-282; Ishida, et al., (1996) Nature Biotechnology14:745-750). Explants are typically transferred to a fresh supply of thesame medium and cultured routinely. A general description of thetechniques and methods for generating transgenic plants are found inAyres and Park, (1994) Critical Reviews in Plant Science 13:219-239 andBommineni and Jauhar, (1997) Maydica 42:107-120. Since the transformedmaterial contains many cells; both transformed and non-transformed cellsare present in any piece of subjected target callus or tissue or groupof cells. The ability to kill non-transformed cells and allowtransformed cells to proliferate results in transformed plant cultures.Often, the ability to remove non-transformed cells is a limitation torapid recovery of transformed plant cells and successful generation oftransgenic plants.

The cells that have been transformed may be grown into plants inaccordance with conventional ways. See, for example, McCormick, et al.,(1986) Plant Cell Reports 5:81-84. These plants may then be grown, andeither pollinated with the same transformed strain or different strains,and the resulting hybrid having constitutive or inducible expression ofthe desired phenotypic characteristic identified. Two or moregenerations may be grown to ensure that expression of the desiredphenotypic characteristic is stably maintained and inherited and thenseeds harvested to ensure that expression of the desired phenotypiccharacteristic has been achieved.

The nucleotide sequences of the embodiments may be provided to the plantby contacting the plant with a virus or viral nucleic acids. Generally,such methods involve incorporating the nucleotide construct of interestwithin a viral DNA or RNA molecule. It is recognized that the disclosedembodiments include IRDIG35563 polypeptides that are initiallysynthesized as part of a viral polyprotein, which later may be processedby proteolysis in vivo or in vitro to produce the desired IRDIG35563polypeptide end product. It is also recognized that such a viralpolyprotein, comprising at least a portion of the amino acid sequence ofan IRDIG35563 of the embodiments, may have the desired pesticidalactivity. Such viral polyproteins and the nucleotide sequences thatencode for them are encompassed by the embodiments. Methods forproviding plants with nucleotide constructs and producing the encodedproteins in the plants, which involve viral DNA or RNA molecules, areknown in the art. See, for example, U.S. Pat. Nos. 5,889,191; 5,889,190;5,866,785; 5,589,367 and 5,316,931; herein incorporated by reference.

Methods for transformation of chloroplasts can be found for example inSvab, et al., (1990) Proc. Natl. Acad. Sci. USA 87:8526-8530; Svab andMaliga, (1993) Proc. Natl. Acad. Sci. USA 90:913-917; Svab and Maliga,(1993) EMBO J. 12:601-606. The method relies on particle gun delivery ofDNA containing a selectable marker and targeting of the DNA to theplastid genome through homologous recombination. Additionally, plastidtransformation can be accomplished by transactivation of a silentplastid-borne transgene by tissue-preferred expression of anuclear-encoded and plastid-directed RNA polymerase. Such a system hasbeen reported in McBride, et al., (1994) Proc. Natl. Acad. Sci. USA91:7301-7305.

The embodiments further relate to plant-propagating material of atransformed plant of the embodiments including, but not limited to,seeds, tubers, corms, bulbs, leaves and cuttings of roots and shoots.

The embodiments may be used for transformation of any plant species,including, but not limited to, monocots and dicots. Examples of plantsof interest include, but are not limited to, corn (Zea mays), Brassicasp. (e.g., B. napus, B. rapa, B. juncea), particularly those Brassicaspecies useful as sources of seed oil, alfalfa (Medicago sativa), rice(Oryza sativa), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghumvulgare), millet (e.g., pearl millet (Pennisetum glaucum), proso millet(Panicum miliaceum), foxtail millet (Setaria italica), finger millet(Eleusine coracana)), sunflower (Helianthus annuus), safflower(Carthamus tinctorius), wheat (Triticum aestivum), soybean (Glycinemax), tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanuts(Arachis hypogaea), cotton (Gossypium barbadense, Gossypium hirsutum),sweet potato (Ipomoea batatus), cassava (Manihot esculenta), coffee(Coffea spp.), coconut (Cocos nucifera), pineapple (Ananas comosus),citrus trees (Citrus spp.), cocoa (Theobroma cacao), tea (Camelliasinensis), banana (Musa spp.), avocado (Persea americana), fig (Ficuscasica), guava (Psidium guajava), mango (Mangifera indica), olive (Oleaeuropaea), papaya (Carica papaya), cashew (Anacardium occidentale),macadamia (Macadamia integrifolia), almond (Prunus amygdalus), sugarbeets (Beta vulgaris), sugarcane (Saccharum spp.), oats, barley,vegetables ornamentals, and conifers.

Vegetables include tomatoes (Lycopersicon esculentum), lettuce (e.g.,Lactuca sativa), green beans (Phaseolus vulgaris), lima beans (Phaseoluslimensis), peas (Lathyrus spp.), and members of the genus Cucumis suchas cucumber (C. sativus), cantaloupe (C. cantalupensis), and musk melon(C. melo). Ornamentals include azalea (Rhododendron spp.), hydrangea(Macrophylla hydrangea), hibiscus (Hibiscus rosasanensis), roses (Rosaspp.), tulips (Tulipa spp.), daffodils (Narcissus spp.), petunias(Petunia hybrida), carnation (Dianthus caryophyllus), poinsettia(Euphorbia pulcherrima), and chrysanthemum. Conifers that may beemployed in practicing the embodiments include, for example, pines suchas loblolly pine (Pinus taeda), slash pine (Pinus elliotii), ponderosapine (Pinus ponderosa), lodgepole pine (Pinus contorta), and Montereypine (Pinus radiata); Douglas-fir (Pseudotsuga menziesii); Westernhemlock (Tsuga canadensis); Sitka spruce (Picea glauca); redwood(Sequoia sempervirens); true firs such as silver fir (Abies amabilis)and balsam fir (Abies balsamea); and cedars such as Western red cedar(Thuja plicata) and Alaska yellow-cedar (Chamaecyparis nootkatensis).Plants of the embodiments include crop plants (for example, corn,alfalfa, sunflower, Brassica, soybean, cotton, safflower, peanut,sorghum, wheat, millet, tobacco, etc.), such as corn and soybean plants.

Turf grasses include, but are not limited to: annual bluegrass (Poaannua); annual ryegrass (Lolium multiflorum); Canada bluegrass (Poacompressa); Chewing's fescue (Festuca rubra); colonial bentgrass(Agrostis tenuis); creeping bentgrass (Agrostis palustris); crestedwheatgrass (Agropyron desertorum); fairway wheatgrass (Agropyroncristatum); hard fescue (Festuca longifolia); Kentucky bluegrass (Poapratensis); orchardgrass (Dactylis glomerata); perennial ryegrass(Lolium perenne); red fescue (Festuca rubra); redtop (Agrostis alba);rough bluegrass (Poa trivialis); sheep fescue (Festuca ovina); smoothbromegrass (Bromus inermis); tall fescue (Festuca arundinacea); timothy(Phleum pratense); velvet bentgrass (Agrostis canina); weepingalkaligrass (Puccinellia distans); western wheatgrass (Agropyronsmithii); Bermuda grass (Cynodon spp.); St. Augustine grass(Stenotaphrum secundatum); zoysia grass (Zoysia spp.); Bahia grass(Paspalum notatum); carpet grass (Axonopus affinis); centipede grass(Eremochloa ophiuroides); kikuyu grass (Pennisetum clandesinum);seashore paspalum (Paspalum vaginatum); blue gramma (Boutelouagracilis); buffalo grass (Buchloe dactyloids); sideoats gramma(Bouteloua curtipendula).

Plants of interest include grain plants that provide seeds of interest,oil-seed plants, and leguminous plants. Seeds of interest include grainseeds, such as corn, wheat, barley, rice, sorghum, rye, millet, etc.Oil-seed plants include cotton, soybean, safflower, sunflower, Brassica,maize, alfalfa, palm, coconut, flax, castor, olive, etc. Leguminousplants include beans and peas. Beans include guar, locust bean,fenugreek, soybean, garden beans, cowpea, mung bean, lima bean, favabean, lentils, chickpea, etc.

Evaluation of Plant Transformation. Following introduction ofheterologous foreign DNA into plant cells, the transformation orintegration of heterologous gene in the plant genome is confirmed byvarious methods such as analysis of nucleic acids, proteins andmetabolites associated with the integrated gene.

PCR analysis is a rapid method to screen transformed cells, tissue orshoots for the presence of incorporated gene at the earlier stage beforetransplanting into the soil (Sambrook and Russell, (2001) MolecularCloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.). PCR is carried out using oligonucleotide primersspecific to the gene of interest or Agrobacterium vector background,etc.

Plant transformation may be confirmed by Southern blot analysis ofgenomic DNA (Sambrook and Russell, (2001) supra). In general, total DNAis extracted from the transformant, digested with appropriaterestriction enzymes, fractionated in an agarose gel and transferred to anitrocellulose or nylon membrane. The membrane or “blot” is then probedwith, for example, radiolabeled 32P target DNA fragment to confirm theintegration of introduced gene into the plant genome according tostandard techniques (Sambrook and Russell, (2001) supra).

In Northern blot analysis, RNA is isolated from specific tissues oftransformant, fractionated in a formaldehyde agarose gel, and blottedonto a nylon filter according to standard procedures that can be foundin Sambrook and Russell, (2001) supra). Expression of RNA encoded by thepesticidal gene is then tested by hybridizing the filter to aradioactive probe derived from a pesticidal gene, by methods known inthe art (Sambrook and Russell, (2001) supra).

Western blot, biochemical assays and the like may be carried out on thetransgenic plants to confirm the presence of protein encoded by thepesticidal gene by standard procedures (Sambrook and Russell, 2001,supra) using antibodies that bind to one or more epitopes present on theIRDIG35563 polypeptide.

Insect Bioassays of transgenic Arabidopsis. Transgenic Arabidopsis linesexpressing modified IRDIG35563 proteins can be used to demonstrateactivity against sensitive insect species in artificial diet overlayassays. Protein extracted from transgenic and non-transgenic Arabidopsislines may be quantified by appropriate methods and sample volumesadjusted to normalize protein concentration. Bioassays are thenconducted on artificial diet as described below. Non-transgenicArabidopsis and/or buffer and water should be included in assays asbackground check treatments.

Bioassay of transgenic maize. Bioactivity of the IRDIG35563 toxins andvariants produced in plant cells also may be demonstrated byconventional bioassay methods (see, for example Huang et al., 2006).Efficacy may be tested by feeding various plant tissues or tissue piecesderived from a plant producing an IRDIG35563 toxin to target insects ina controlled feeding environment. Alternatively, protein extracts may beprepared from various plant tissues derived from a plant producing theIRDIG35563 toxin and incorporate the extracted proteins in an artificialdiet bioassay. It is to be understood that the results of such feedingassays are to be compared to similarly conducted bioassays that employappropriate control tissues from host plants that do not produce theIRDIG35563 protein or variants, or to other control samples.

Methods to Introduce Genome Editing Technologies into Plants. In someembodiments, the disclosed IRDIG35563 polynucleotide compositions can beintroduced into the genome of a plant using genome editing technologies,or previously introduced IRDIG35563 polynucleotides in the genome of aplant may be edited using genome editing technologies. For example, thedisclosed polynucleotides can be introduced into a desired location inthe genome of a plant through the use of double-stranded breaktechnologies such as TALENs, meganucleases, zinc finger nucleases,CRISPR-Cas, and the like. For example, the disclosed polynucleotides canbe introduced into a desired location in a genome using a CRISPR-Cassystem, for the purpose of site-specific insertion. The desired locationin a plant genome can be any desired target site for insertion, such asa genomic region amenable for breeding or may be a target site locatedin a genomic window with an existing trait of interest. Existing traitsof interest could be either an endogenous trait or a previouslyintroduced trait.

In some embodiments, where the disclosed IRDIG35563 polynucleotide haspreviously been introduced into a genome, genome editing technologiesmay be used to alter or modify the introduced polynucleotide sequence.Site specific modifications that can be introduced into the disclosedIRDIG35563 polynucleotide compositions include those produced using anymethod for introducing site specific modification, including, but notlimited to, through the use of gene repair oligonucleotides (e.g. USPublication 2013/0019349), or through the use of double-stranded breaktechnologies such as TALENs, meganucleases, zinc finger nucleases,CRISPR-Cas, and the like. Such technologies can be used to modify thepreviously introduced polynucleotide through the insertion, deletion orsubstitution of nucleotides within the introduced polynucleotide.Alternatively, double-stranded break technologies can be used to addadditional nucleotide sequences to the introduced polynucleotide.Additional sequences that may be added include, additional expressionelements, such as enhancer and promoter sequences. In anotherembodiment, genome editing technologies may be used to positionadditional insecticidally-active proteins in close proximity to thedisclosed IRDIG35563 polynucleotide compositions disclosed herein withinthe genome of a plant, to generate molecular stacks ofinsecticidally-active proteins.

An “altered target site,” “altered target sequence.” “modified targetsite,” and “modified target sequence” are used interchangeably hereinand refer to a target sequence as disclosed herein that comprises atleast one alteration when compared to non-altered target sequence. Such“alterations” include, for example: (i) replacement of at least onenucleotide, (ii) a deletion of at least one nucleotide, (iii) aninsertion of at least one nucleotide, or (iv) any combination of(i)-(iii).

Stacking of Traits in Transgenic Plants. Transgenic plants may comprisea stack of one or more insecticidal polynucleotides disclosed hereinwith one or more additional polynucleotides resulting in the productionor suppression of multiple polypeptide sequences. Transgenic plantscomprising stacks of polynucleotide sequences can be obtained by eitheror both of traditional breeding methods or through genetic engineeringmethods. These methods include, but are not limited to, breedingindividual lines each comprising a polynucleotide of interest,transforming a transgenic plant comprising a gene disclosed herein witha subsequent gene and co-transformation of genes into a single plantcell. As used herein, the term “stacked” includes having the multipletraits present in the same plant (i.e., both traits are incorporatedinto the nuclear genome, one trait is incorporated into the nucleargenome and one trait is incorporated into the genome of a plastid orboth traits are incorporated into the genome of a plastid). In onenon-limiting example, “stacked traits” comprise a molecular stack wherethe sequences are physically adjacent to each other. A trait, as usedherein, refers to the phenotype derived from a particular sequence orgroups of sequences. Co-transformation of genes can be carried out usingsingle transformation vectors comprising multiple genes or genes carriedseparately on multiple vectors. If the sequences are stacked bygenetically transforming the plants, the polynucleotide sequences ofinterest can be combined at any time and in any order. The traits can beintroduced simultaneously in a co-transformation protocol with thepolynucleotides of interest provided by any combination oftransformation cassettes. For example, if two sequences will beintroduced, the two sequences can be contained in separatetransformation cassettes (trans) or contained on the same transformationcassette (cis). Expression of the sequences can be driven by the samepromoter or by different promoters. In certain cases, it may bedesirable to introduce a transformation cassette that will suppress theexpression of the polynucleotide of interest. This may be combined withany combination of other suppression cassettes or overexpressioncassettes to generate the desired combination of traits in the plant. Itis further recognized that polynucleotide sequences can be stacked at adesired genomic location using a site-specific recombination system.See, for example, WO 1999/25821, WO 1999/25854, WO 1999/25840, WO1999/25855 and WO 1999/25853, all of which are herein incorporated byreference.

In some embodiments, the polynucleotides encoding the IRDIG35563polypeptide disclosed herein, alone or stacked with one or moreadditional insect resistance traits can be stacked with one or moreadditional input traits (e.g., herbicide resistance, fungal resistance,virus resistance, stress tolerance, disease resistance, male sterility,stalk strength, and the like) or output traits (e.g., increased yield,modified starches, improved oil profile, balanced amino acids, highlysine or methionine, increased digestibility, improved fiber quality,drought resistance, and the like). Thus, the polynucleotide embodimentscan be used to provide a complete agronomic package of improved cropquality with the ability to flexibly and cost effectively control anynumber of agronomic pests.

Transgenes useful for stacking include but are not limited to:

1. Transgenes that Confer Resistance to Insects or Disease and thatEncode:

(A) Plant disease resistance genes. Plant defenses are often activatedby specific interaction between the product of a disease resistance gene(R) in the plant and the product of a corresponding avirulence (Avr)gene in the pathogen. A plant variety can be transformed with clonedresistance gene to engineer plants that are resistant to specificpathogen strains. See, for example, Jones, et al., (1994) Science266:789 (cloning of the tomato Cf-9 gene for resistance to Cladosporiumfulvum); Martin, et al., (1993) Science 262:1432 (tomato Pto gene forresistance to Pseudomonas syringae pv. tomato encodes a protein kinase);Mindrinos, et al., (1994) Cell 78:1089 (Arabidopsis RSP2 gene forresistance to Pseudomonas syringae), McDowell and Woffenden, (2003)Trends Biotechnol. 21(4):178-83 and Toyoda, et al., (2002) TransgenicRes. 11(6):567-82. A plant resistant to a disease is one that is moreresistant to a pathogen as compared to the wild type plant.

(B) Genes encoding a Bacillus thuringiensis protein, a derivativethereof or a synthetic polypeptide modeled thereon. See, for example,Geiser, et al., (1986) Gene 48:109, who disclose the cloning andnucleotide sequence of a Bt delta-endotoxin gene. Moreover, DNAmolecules encoding delta-endotoxin genes can be purchased from AmericanType Culture Collection (Rockville, Md.), for example, under ATCC®Accession Numbers 40098, 67136, 31995 and 31998. Other non-limitingexamples of Bacillus thuringiensis transgenes being geneticallyengineered are given in the following patents and patent applicationsand hereby are incorporated by reference for this purpose: U.S. Pat.Nos. 5,188,960; 5,689,052; 5,880,275; 5,986,177; 6,023,013, 6,060,594,6,063,597, 6,077,824, 6,620,988, 6,642,030, 6,713,259, 6,893,826,7,105,332; 7,179,965, 7,208,474; 7,227,056, 7,288,643, 7,323,556,7,329,736, 7,449,552, 7,468,278, 7,510,878, 7,521,235, 7,544,862,7,605,304, 7,696,412, 7,629,504, 7,705,216, 7,772,465, 7,790,846,7,858,849 and WO 1991/14778; WO 1999/31248; WO 2001/12731; WO 1999/24581and WO 1997/40162.

Genes encoding pesticidal proteins may also be stacked including, butare not limited to: insecticidal proteins from Pseudomonas sp. such asPSEEN3174 (Monalysin; (2011) PLoS Pathogens 7:1-13); from Pseudomonasprotegens strain CHAO and Pf-5 (previously fluorescens) (Pechy-Tarr,(2008) Environmental Microbiology 10:2368-2386; GenBank Accession No.EU400157); from Pseudomonas taiwanensis (Liu, et al., (2010) J. Agric.Food Chem., 58:12343-12349) and from Pseudomonas pseudoalcaligenes(Zhang, et al., (2009) Annals of Microbiology 59:45-50 and Li, et al.,(2007) Plant Cell Tiss. Organ Cult. 89:159-168); insecticidal proteinsfrom Photorhabdus sp. and Xenorhabdus sp. (Hinchliffe, et al., (2010)The Open Toxicology Journal, 3:101-118 and Morgan, et al., (2001)Applied and Envir. Micro. 67:2062-2069); U.S. Pat. Nos. 6,048,838, and6,379,946; a PIP-1 polypeptide of U.S. Pat. No. 9,688,730; an AfIP-1Aand/or AfIP-1B polypeptide of U.S. Pat. No. 9,475,847; a PIP-47polypeptide of US Publication Number US20160186204; an IPD045polypeptide, an IPDO64 polypeptide, an IPD074 polypeptide, an IPD075polypeptide, and an IPD077 polypeptide of PCT Publication Number WO2016/114973; an IPDO80 polypeptide of PCT Serial Number PCT/US17/56517;an IPD078 polypeptide, an IPDO84 polypeptide, an IPD085 polypeptide, anIPD086 polypeptide, an IPD087 polypeptide, an IPDO88 polypeptide, and anIPD089 polypeptide of Serial Number PCT/US17/54160; PIP-72 polypeptideof US Patent Publication Number US20160366891; a PtIP-50 polypeptide anda PtIP-65 polypeptide of US Publication Number US20170166921; an IPDO98polypeptide, an IPD059 polypeptide, an IPD108 polypeptide, an IPD109polypeptide of U.S. Ser. No. 62/521,084; a PtIP-83 polypeptide of USPublication Number US20160347799; a PtIP-96 polypeptide of USPublication Number US20170233440; an IPD079 polypeptide of PCTPublication Number WO2017/23486; an IPD082 polypeptide of PCTPublication Number WO 2017/105987, an IPDO90 polypeptide of SerialNumber PCT/US17/30602, an IPDO93 polypeptide of U.S. Ser. No.62/434,020; an IPD103 polypeptide of Serial Number PCT/US17/39376; anIPD101 polypeptide of U.S. Ser. No. 62/438,179; an IPD121 polypeptide ofUS Serial Number U.S. 62/508,514; and δ-endotoxins including, but notlimited to a Cry1, Cry2, Cry3, Cry4, Cry5, Cry6, Cry7, Cry8, Cry9,Cry10, Cry11, Cry12, Cry13, Cry14, Cry15, Cry16, Cry17, Cry18, Cry19,Cry20, Cry21, Cry22, Cry23, Cry24, Cry25, Cry26, Cry27, Cry28, Cry29,Cry30, Cry31, Cry32, Cry33, Cry34, Cry35, Cry36, Cry37, Cry38, Cry39,Cry40, Cry41, Cry42, Cry43, Cry44, Cry45, Cry46, Cry47, Cry49, Cry50,Cry51, Cry52, Cry53, Cry54, Cry55, Cry56, Cry57, Cry58, Cry59, Cry60,Cry61, Cry62, Cry63, Cry64, Cry65, Cry66, Cry67, Cry68, Cry69, Cry70,Cry71, and Cry 72 classes of δ-endotoxin polypeptides and the B.thuringiensis cytolytic cyt1 and cyt2 genes. Members of these classes ofB. thuringiensis insecticidal proteins can be found in Crickmore, etal., “Bacillus thuringiensis toxin nomenclature” (2011), atlifesci.sussex.ac.uk/home/Neil_Crickmore/Bt/ which can be accessed onthe world-wide web using the “www” prefix).

Examples of δ-endotoxins also include but are not limited to Cry1Aproteins of U.S. Pat. Nos. 5,880,275, 7,858,849, and 8,878,007; a Cry1Acmutant of U.S. Pat. No. 9,512,187; a DIG-3 or DIG-11 toxin (N-terminaldeletion of α-helix 1 and/or α-helix 2 variants of cry proteins such asCry1A, Cry3A) of U.S. Pat. Nos. 8,304,604, 8,304,605 and 8,476,226;Cry1B of U.S. patent application Ser. No. 10/525,318, US PatentApplication Publication Number US20160194364, and U.S. Pat. Nos.9,404,121 and 8,772,577; Cry1B variants of PCT Publication NumberWO2016/61197 and Serial Number PCT/US17/27160; Cry1C of U.S. Pat. No.6,033,874; Cry1D protein of US20170233759; a Cry1E protein of PCT SerialNumber PCT/US17/53178; a Cry1F protein of U.S. Pat. Nos. 5,188,960 and6,218,188; Cry1A/F chimeras of U.S. Pat. Nos. 7,070,982; 6,962,705 and6,713,063; a Cry1I protein of PCT Publication number WO 2017/0233759; aCry1J variant of US Publication US20170240603; a Cry2 protein such asCry2Ab protein of U.S. Pat. No. 7,064,249 and Cry2A.127 protein of U.S.Pat. No. 7,208,474; a Cry3A protein including but not limited to anengineered hybrid insecticidal protein (eHIP) created by fusing uniquecombinations of variable regions and conserved blocks of at least twodifferent Cry proteins (US Patent Application Publication Number2010/0017914); a Cry4 protein; a Cry5 protein; a Cry6 protein; Cry8proteins of U.S. Pat. Nos. 7,329,736, 7,449,552, 7,803,943, 7,476,781,7,105,332, 7,339,092, 7,378,499, 7,462,760, and 9,593,345; a Cry9protein such as such as members of the Cry9A, Cry9B, Cry9C, Cry9D, Cry9Eand Cry9F families including the Cry9 protein of U.S. Pat. Nos.9,000,261 and 8,802,933, and US Serial Number WO 2017/132188; a Cry15protein of Naimov, et al., (2008) Applied and EnvironmentalMicrobiology, 74:7145-7151; a Cry14 protein of U.S. Pat. No. 8,933,299;a Cry22, a Cry34Ab1 protein of U.S. Pat. Nos. 6,127,180, 6,624,145 and6,340,593; a truncated Cry34 protein of U.S. Pat. No. 8,816,157; aCryET33 and cryET34 protein of U.S. Pat. Nos. 6,248,535, 6,326,351,6,399,330, 6,949,626, 7,385,107 and 7,504,229; a CryET33 and CryET34homologs of US Patent Publication Number 2006/0191034, 2012/0278954, andPCT Publication Number WO 2012/139004; a Cry35Ab1 protein of U.S. Pat.Nos. 6,083,499, 6,548,291 and 6,340,593; a Cry46 protein of U.S. Pat.No. 9,403,881, a Cry 51 protein, a Cry binary toxin; a TIC901 or relatedtoxin; TIC807 of US Patent Application Publication Number 2008/0295207;TIC853 of U.S. Pat. No. 8,513,493; ET29, ET37, TIC809, TIC810, TIC812,TIC127, TIC128 of PCT US 2006/033867; engineered Hemipteran toxicproteins of US Patent Application Publication Number US20160150795,AXMI-027, AXMI-036, and AXMI-038 of U.S. Pat. No. 8,236,757; AXMI-031,AXMI-039, AXMI-040, AXMI-049 of U.S. Pat. No. 7,923,602; AXMI-018,AXMI-020 and AXMI-021 of WO 2006/083891; AXMI-010 of WO 2005/038032;AXMI-003 of WO 2005/021585; AXMI-008 of US Patent ApplicationPublication Number 2004/0250311; AXMI-006 of US Patent ApplicationPublication Number 2004/0216186; AXMI-007 of US Patent ApplicationPublication Number 2004/0210965; AXMI-009 of US Patent ApplicationNumber 2004/0210964; AXMI-014 of US Patent Application PublicationNumber 2004/0197917; AXMI-004 of US Patent Application PublicationNumber 2004/0197916; AXMI-028 and AXMI-029 of WO 2006/119457; AXMI-007,AXMI-008, AXMI-0080rf2, AXMI-009, AXMI-014 and AXMI-004 of WO2004/074462; AXMI-150 of U.S. Pat. No. 8,084,416; AXMI-205 of US PatentApplication Publication Number 2011/0023184; AXMI-011, AXMI-012,AXMI-013, AXMI-015, AXMI-019, AXMI-044, AXMI-037, AXMI-043, AXMI-033,AXMI-034, AXMI-022, AXMI-023, AXMI-041, AXMI-063 and AXMI-064 of USPatent Application Publication Number 2011/0263488; AXMI046, AXMI048,AXMI050, AXMI051, AXMI052, AXMI053, AXMI054, AXMI055, AXMI056, AXMI057,AXMI058, AXMI059, AXMI060, AXMI061, AXMI067, AXMI069, AXMI071, AXMI072,AXMI073, AXMI074, AXMI075, AXMI087, AXMI088, AXMI093, AXMI070, AXMI080,AXMI081, AXMI082, AXMI091, AXMI092, AXMI096, AXMI097, AXMI098, AXMI099,AXMI100, AXMI101, AXMI102, AXMI103, AXMI104, AXMI107, AXMI108, AXMI109,AXMI110, AXMI111, AXMI112, AXMI114, AXMI116, AXMI117, AXMI118, AXMI119,AXMI120, AXMI121, AXMI122, AXMI123, AXMI124, AXMI125, AXMI126, AXMI127,AXMI129, AXMI151, AXMI161, AXMI164, AXMI183, AXMI132, AXMI137, AXMI138of U.S. Pat. Nos. 8,461,421 and 8,461,422; AXMI-R1 and related proteinsof US Patent Application Publication Number 2010/0197592; AXMI221Z,AXMI222z, AXMI223z, AXMI224z and AXMI225z of WO 2011/103248; AXMI218,AXMI219, AXMI220, AXMI226, AXMI227, AXMI228, AXMI229, AXMI230 andAXMI231 of WO 2011/103247; AXMI-115, AXMI-113, AXMI-005, AXMI-163 andAXMI-184 of U.S. Pat. No. 8,334,431; AXMI-001, AXMI-002, AXMI-030,AXMI-035 and AXMI-045 of US Patent Application Publication Number2010/0298211; AXMI-066 and AXMI-076 of US Patent Application PublicationNumber 2009/0144852; AXMI128, AXMI130, AXMI131, AXMI133, AXMI140,AXMI141, AXMI142, AXMI143, AXMI144, AXMI146, AXMI148, AXMI149, AXMI152,AXMI153, AXMI154, AXMI155, AXMI156, AXMI157, AXMI158, AXMI162, AXMI165,AXMI166, AXMI167, AXMI168, AXMI169, AXMI170, AXMI171, AXMI172, AXMI173,AXMI174, AXMI175, AXMI176, AXMI177, AXMI178, AXMI179, AXMI180, AXMI181,AXMI182, AXMI185, AXMI186, AXMI187, AXMI188, AXMI189 of U.S. Pat. No.8,318,900; AXMI079, AXMI080, AXMI081, AXMI082, AXMI091, AXMI092,AXMI096, AXMI097, AXMI098, AXMI099, AXMI100, AXMI101, AXMI102, AXMI103,AXMI104, AXMI107, AXMI108, AXMI109, AXMI110, dsAXMI111, AXMI112,AXMI114, AXMI116, AXMI117, AXMI118, AXMI119, AXMI120, AXMI121, AXMI122,AXMI123, AXMI124, AXMI1257, AXMI1268, AXMI127, AXMI129, AXMI164,AXMI151, AXMI161, AXMI183, AXMI132, AXMI138, AXMI137 of U.S. Pat. No.8,461,421; AXMI192 of U.S. Pat. No. 8,461,415; AXMI281 of US PatentApplication Publication Number US20160177332; AXMI422 of U.S. Pat. No.8,252,872; cry proteins such as Cry1A and Cry3A having modifiedproteolytic sites of U.S. Pat. No. 8,319,019; a Cry1Ac, Cry2Aa andCry1Ca toxin protein from Bacillus thuringiensis strain VBTS 2528 of USPatent Application Publication Number 2011/0064710. The Cry proteinsMP032, MP049, MP051, MP066, MP068, MP070, MP091S, MP109S, MP114, MP121,MP134S, MP183S, MP185S, MP186S, MP195S, MP197S, MP208S, MP209S, MP212S,MP214S, MP217S, MP222S, MP234S, MP235S, MP237S, MP242S, MP243, MP248,MP249S, MP251M, MP252S, MP253, MP259S, MP287S, MP288S, MP295S, MP296S,MP297S, MP300S, MP304S, MP306S, MP310S, MP312S, MP314S, MP319S, MP325S,MP326S, MP327S, MP328S, MP334S, MP337S, MP342S, MP349S, MP356S, MP359S,MP360S, MP437S, MP451S, MP452S, MP466S, MP468S, MP476S, MP482S, MP522S,MP529S, MP548S, MP552S, MP562S, MP564S, MP566S, MP567S, MP569S, MP573S,MP574S, MP575S, MP581S, MP590, MP594S, MP596S, MP597, MP599S, MP600S,MP601S, MP602S, MP604S, MP626S, MP629S, MP630S, MP631S, MP632S, MP633S,MP634S, MP635S, MP639S, MP640S, MP644S, MP649S, MP651S, MP652S, MP653S,MP661S, MP666S, MP672S, MP696S, MP704S, MP724S, MP729S, MP739S, MP755S,MP773S, MP799S, MP800S, MP801S, MP802S, MP803S, MP805S, MP809S, MP815S,MP828S, MP831S, MP844S, MP852, MP865S, MP879S, MP887S, MP891S, MP896S,MP898S, MP935S, MP968, MP989, MP993, MP997, MP1049, MP1066, MP1067,MP1080, MP1081, MP1200, MP1206, MP1233, and MP1311 of U.S. Ser. No.62/607,372. The insecticidal activity of Cry proteins can be found forexample in van Frannkenhuyzen, (2009) J. Invert. Path. 101:1-16). Theuse of Cry proteins as transgenic plant traits art and Cry-transgenicplants including but not limited to plants expressing Cry1Ac,Cry1Ac+Cry2Ab, Cry1Ab, Cry1A.105, Cry1F, Cry1Fa2, Cry1F+Cry1Ac, Cry2Ab,Cry3A, mCry3A, Cry3Bb1, Cry34Ab1, Cry35Ab1, Vip3A, mCry3A, Cry9c andCBI-Bt have received regulatory approval (see, Sanahuja, (2011) PlantBiotech Journal 9:283-300 and the CERA. (2010) GM Crop Database Centerfor Environmental Risk Assessment (CERA), ILSI Research Foundation,Washington D.C. at cera-gmc.org/index.php?action=gm_crop_database whichcan be accessed on the world-wide web using the “www” prefix). More thanone pesticidal proteins can also be expressed in plants such as Vip3Ab &Cry1Fa (US2012/0317682); Cry1BE & CryIF (US2012/0311746); Cry1CA &Cry1AB (US2012/0311745); Cry1F & CryCa (US2012/0317681); Cry1DA & Cry1BE(US2012/0331590); Cry1DA & Cry1Fa (US2012/0331589); Cry1AB & Cry1BE(US2012/0324606); Cry1Fa & Cry2Aa and Cry1I & Cry1E (US2012/0324605);Cry34Ab/35Ab & Cry6Aa (US20130167269); Cry34Ab/VCry35Ab & Cry3Aa(US20130167268); Cry1 Da & Cry1Ca (U.S. Pat. No. 9,796,982); Cry3Aa &Cry6Aa (U.S. Pat. No. 9,798,963); and Cry3A & Cry1Ab or Vip3Aa (U.S.Pat. No. 9,045,766). Pesticidal proteins also include insecticidallipases including lipid acyl hydrolases of U.S. Pat. No. 7,491,869, andcholesterol oxidases such as from Streptomyces (Purcell et al. (1993)Biochem Biophys Res Commun 15:1406-1413). Pesticidal proteins alsoinclude VIP (vegetative insecticidal proteins) toxins of U.S. Pat. Nos.5,877,012, 6,107,279 6,137,033, 7,244,820, 7,615,686, and 8,237,020 andthe like. Other VIP proteins can be found atlifesci.sussex.ac.uk/home/Neil_Crickmore/Bt/vip.html, which can beaccessed on the world-wide web using the “www” prefix. Pesticidalproteins also include Cyt proteins including Cyt1A variants of PCTSerial Number PCT/US2017/000510; Pesticidal proteins also include toxincomplex (TC) proteins, obtainable from organisms such as Xenorhabdus,Photorhabdus and Paenibacillus (see, U.S. Pat. Nos. 7,491,698 and8,084,418). Some TC proteins have “stand alone” insecticidal activityand other TC proteins enhance the activity of the stand-alone toxinsproduced by the same given organism. The toxicity of a “stand-alone” TCprotein (from Photorhabdus, Xenorhabdus or Paenibacillus, for example)can be enhanced by one or more TC protein “potentiators” derived from asource organism of a different genus. There are three main types of TCproteins. As referred to herein, Class A proteins (“Protein A”) arestand-alone toxins. Class B proteins (“Protein B”) and Class C proteins(“Protein C”) enhance the toxicity of Class A proteins. Examples ofClass A proteins are TcbA, TcdA, XptA1 and XptA2. Examples of Class Bproteins are TcaC, TcdB, XptB1Xb and XptC1Wi. Examples of Class Cproteins are TccC, XptC1Xb and XptB1Wi. Pesticidal proteins also includespider, snake and scorpion venom proteins. Examples of spider venompeptides include but not limited to lycotoxin-1 peptides and mutantsthereof (U.S. Pat. No. 8,334,366).

(C) A polynucleotide encoding an insect-specific hormone or pheromonesuch as an ecdysteroid and juvenile hormone, a variant thereof, amimetic based thereon or an antagonist or agonist thereof. See, forexample, the disclosure by Hammock, et al., (1990) Nature 344:458, ofbaculovirus expression of cloned juvenile hormone esterase, aninactivator of juvenile hormone.

(D) A polynucleotide encoding an insect-specific peptide which, uponexpression, disrupts the physiology of the affected pest. For example,see the disclosures of, Regan, (1994) J. Biol. Chem. 269:9 (expressioncloning yields DNA coding for insect diuretic hormone receptor); Pratt,et al., (1989) Biochem. Biophys. Res. Comm. 163:1243 (an allostatin isidentified in Diploptera puntata); Chattopadhyay, et al., (2004)Critical Reviews in Microbiology 30(1):33-54; Zjawiony, (2004) J NatProd 67(2):300-310; Carlini and Grossi-de-Sa, (2002) Toxicon40(11):1515-1539; Ussuf, et al., (2001) Curr Sci. 80(7):847-853 andVasconcelos and Oliveira, (2004) Toxicon 44(4):385-403. See also, U.S.Pat. No. 5,266,317 to Tomalski, et al., who disclose genes encodinginsect-specific toxins.

(E) A polynucleotide encoding an enzyme responsible for ahyperaccumulation of a monoterpene, a sesquiterpene, a steroid,hydroxamic acid, a phenylpropanoid derivative or another non-proteinmolecule with insecticidal activity.

(F) A polynucleotide encoding an enzyme involved in the modification,including the post-translational modification, of a biologically activemolecule; for example, a glycolytic enzyme, a proteolytic enzyme, alipolytic enzyme, a nuclease, a cyclase, a transaminase, an esterase, ahydrolase, a phosphatase, a kinase, a phosphorylase, a polymerase, anelastase, a chitinase and a glucanase, whether natural or synthetic.See, PCT Application WO 1993/02197 in the name of Scott, et al., whichdiscloses the nucleotide sequence of a callase gene. DNA molecules whichcontain chitinase-encoding sequences can be obtained, for example, fromthe ATCC® under Accession Numbers 39637 and 67152. See also, Kramer, etal., (1993) Insect Biochem. Molec. Biol. 23:691, who teach thenucleotide sequence of a cDNA encoding tobacco hookworm chitinase andKawalleck, et al., (1993) Plant Molec. Biol. 21:673, who provide thenucleotide sequence of the parsley ubi4-2 polyubiquitin gene, and U.S.Pat. Nos. 6,563,020; 7,145,060 and 7,087,810.

(G) A polynucleotide encoding a molecule that stimulates signaltransduction. For example, see the disclosure by Botella, et al., (1994)Plant Molec. Biol. 24:757, of nucleotide sequences for mung beancalmodulin cDNA clones, and Griess, et al., (1994) Plant Physiol.104:1467, who provide the nucleotide sequence of a maize calmodulin cDNAclone.

(H) A polynucleotide encoding a hydrophobic moment peptide. See, PCTApplication WO 1995/16776 and U.S. Pat. No. 5,580,852 disclosure ofpeptide derivatives of Tachyplesin which inhibit fungal plant pathogens)and PCT Application WO 1995/18855 and U.S. Pat. No. 5,607,914 (teachessynthetic antimicrobial peptides that confer disease resistance).

(I) A polynucleotide encoding a membrane permease, a channel former or achannel blocker. For example, see the disclosure by Jaynes, et al.,(1993) Plant Sci. 89:43, of heterologous expression of a cecropin-betalytic peptide analog to render transgenic tobacco plants resistant toPseudomonas solanacearum.

(J) A gene encoding a viral-invasive protein or a complex toxin derivedtherefrom. For example, the accumulation of viral coat proteins intransformed plant cells imparts resistance to viral infection and/ordisease development effected by the virus from which the coat proteingene is derived, as well as by related viruses. See, Beachy, et al.,(1990) Ann. Rev. Phytopathol. 28:451. Coat protein-mediated resistancehas been conferred upon transformed plants against alfalfa mosaic virus,cucumber mosaic virus, tobacco streak virus, potato virus X, potatovirus Y, tobacco etch virus, tobacco rattle virus and tobacco mosaicvirus. Id.

(K) A gene encoding an insect-specific antibody or an immunotoxinderived therefrom. Thus, an antibody targeted to a critical metabolicfunction in the insect gut would inactivate an affected enzyme, killingthe insect. Cf. Taylor, et al., Abstract #497, SEVENTH INT'L SYMPOSIUMON MOLECULAR PLANT-MICROBE INTERACTIONS (Edinburgh, Scotland, 1994)(enzymatic inactivation in transgenic tobacco via production ofsingle-chain antibody fragments).

(L) A gene encoding a virus-specific antibody. See, for example,Tavladoraki, et al., (1993) Nature 366:469, who show that transgenicplants expressing recombinant antibody genes are protected from virusattack.

(M) A polynucleotide encoding a developmental-arrestive protein producedin nature by a pathogen or a parasite. Thus, fungal endoalpha-1,4-D-polygalacturonases facilitate fungal colonization and plantnutrient release by solubilizing plant cell wallhomo-alpha-1,4-D-galacturonase. See, Lamb, et al., (1992) Bio Technology10:1436. The cloning and characterization of a gene which encodes a beanendopolygalacturonase-inhibiting protein is described by Toubart, etal., (1992) Plant J. 2:367.

(N) A polynucleotide encoding a developmental-arrestive protein producedin nature by a plant. For example, Logemann, et al., (1992) BioTechnology 10:305, have shown that transgenic plants expressing thebarley ribosome-inactivating gene have an increased resistance to fungaldisease.

(O) Genes involved in the Systemic Acquired Resistance (SAR) Responseand/or the pathogenesis related genes. Briggs, (1995) Current Biology5(2), Pieterse and Van Loon, (2004) Curr. Opin. Plant Bio. 7(4):456-64and Somssich, (2003) Cell 113(7):815-6.

(P) Antifungal genes (Cornelissen and Melchers, (1993) Pl. Physiol.101:709-712 and Parijs, et al., (1991) Planta 183:258-264 and Bushnell,et al., (1998) Can. J. of Plant Path. 20(2):137-149. Also see, U.S.patent application Ser. Nos. 09/950,933; 11/619,645; 11/657,710;11/748,994; 11/774,121 and U.S. Pat. Nos. 6,891,085 and 7,306,946. LysMReceptor-like kinases for the perception of chitin fragments as a firststep in plant defense response against fungal pathogens (US2012/0110696).

(Q) Detoxification genes, such as for fumonisin, beauvericin,moniliformin and zearalenone and their structurally related derivatives.For example, see, U.S. Pat. Nos. 5,716,820; 5,792,931; 5,798,255;5,846,812; 6,083,736; 6,538,177; 6,388,171 and 6,812,380.

(R) A polynucleotide encoding a Cystatin and cysteine proteinaseinhibitors. See, U.S. Pat. No. 7,205,453.

(S) Defensin genes. See, WO 2003/000863 and U.S. Pat. Nos. 6,911,577;6,855,865; 6,777,592 and 7,238,781.

(T) Genes conferring resistance to nematodes. See, e.g., PCT ApplicationWO 1996/30517; PCT Application WO 1993/19181, WO 2003/033651 and Urwin,et al., (1998) Planta 204:472-479, Williamson, (1999) Curr Opin PlantBio. 2(4):327-31; U.S. Pat. Nos. 6,284,948 and 7,301,069 and miR164genes (WO 2012/058266).

(U) Genes that confer resistance to Phytophthora Root Rot, such as theRps 1, Rps 1-a, Rps 1-b, Rps 1-c, Rps 1-d, Rps 1-e, Rps 1-k, Rps 2, Rps3-a, Rps 3-b, Rps 3-c, Rps 4, Rps 5, Rps 6, Rps 7 and other Rps genes.See, for example, Shoemaker, et al., Phytophthora Root Rot ResistanceGene Mapping in Soybean, Plant Genome IV Conference, San Diego, Calif.(1995).

(V) Genes that confer resistance to Brown Stem Rot, such as described inU.S. Pat. No. 5,689,035 and incorporated by reference for this purpose.

(W) Genes that confer resistance to Colletotrichum, such as described inUS Patent Application Publication US 2009/0035765 and incorporated byreference for this purpose. This includes the Rcg locus that may beutilized as a single locus conversion.

2. Transgenes that Confer Resistance to an Herbicide, for Example:

(A) A polynucleotide encoding resistance to an herbicide that inhibitsthe growing point or meristem, such as an imidazolinone or asulfonylurea. Exemplary genes in this category code for mutant ALS andAHAS enzyme as described, for example, by Lee, et al., (1988) EMBO J.7:1241 and Miki, et al., (1990) Theor. Appl. Genet. 80:449,respectively. See also, U.S. Pat. Nos. 5,605,011; 5,013,659; 5,141,870;5,767,361; 5,731,180; 5,304,732; 4,761,373; 5,331,107; 5,928,937 and5,378,824; U.S. patent application Ser. No. 11/683,737 and InternationalPublication WO 1996/33270.

(B) A polynucleotide encoding a protein for resistance to Glyphosate(resistance imparted by mutant 5-enolpyruvl-3-phosphikimate synthase(EPSP) and aroA genes, respectively) and other phosphono compounds suchas glufosinate (phosphinothricin acetyl transferase (PAT), Streptomycescoelicolor (A3) selectable marker (DSM-2), and Streptomyceshygroscopicus phosphinothricin acetyl transferase (bar) genes), andpyridinoxy or phenoxy proprionic acids and cyclohexones (ACCaseinhibitor-encoding genes). See, for example, U.S. Pat. No. 4,940,835 toShah, et al., which discloses the nucleotide sequence of a form of EPSPSwhich can confer glyphosate resistance. U.S. Pat. No. 5,627,061 toBarry, et al., also describes genes encoding EPSPS enzymes. See also,U.S. Pat. Nos. 6,566,587; 6,338,961; 6,248,876; 6,040,497; 5,804,425;5,633,435; 5,145,783; 4,971,908; 5,312,910; 5,188,642; 5,094,945,4,940,835; 5,866,775; 6,225,114; 6,130,366; 5,310,667; 4,535,060;4,769,061; 5,633,448; 5,510,471; Re. 36,449; RE 37,287 E and 5,491,288and International Publications EP 1173580; WO 2001/66704; EP 1173581 andEP 1173582, which are incorporated herein by reference for this purpose.Glyphosate resistance is also imparted to plants that express a geneencoding a glyphosate oxidoreductase enzyme as described more fully inU.S. Pat. Nos. 5,776,760 and 5,463,175, which are incorporated herein byreference for this purpose. In addition, glyphosate resistance can beimparted to plants by the over expression of genes encoding glyphosateN-acetyltransferase. See, for example, U.S. Pat. Nos. 7,462,481;7,405,074 and US Patent Application Publication Number US 2008/0234130.A DNA molecule encoding a mutant aroA gene can be obtained under ATCC®Accession Number 39256, and the nucleotide sequence of the mutant geneis disclosed in U.S. Pat. No. 4,769,061 to Comai. EP Application Number0 333 033 to Kumada, et al., and U.S. Pat. No. 4,975,374 to Goodman, etal., disclose nucleotide sequences of glutamine synthetase genes whichconfer resistance to herbicides such as L-phosphinothricin. Thenucleotide sequence of a phosphinothricin-acetyl-transferase gene isprovided in EP Application Numbers 0 242 246 and 0 242 236 to Leemans,et al.; De Greef, et al., (1989) Bio Technology 7:61, describe theproduction of transgenic plants that express chimeric bar genes codingfor phosphinothricin acetyl transferase activity. See also, U.S. Pat.Nos. 5,969,213; 5,489,520; 5,550,318; 5,874,265; 5,919,675; 5,561,236;5,648,477; 5,646,024; 6,177,616 and 5,879,903, which are incorporatedherein by reference for this purpose. US 2011/0107455 discloses theDSM-2 gene and gene product sequences as well as their use inglufosinate herbicide-resistant plants. Exemplary genes conferringresistance to phenoxy proprionic acids and cyclohexones, such assethoxydim and haloxyfop, are the Acc1-S1, Acc1-S2 and Acc1-S3 genesdescribed by Marshall, et al., (1992) Theor. Appl. Genet. 83:435.

(C) A polynucleotide encoding a protein for resistance to herbicide thatinhibits photosynthesis, such as a triazine (psbA and gs+ genes) and abenzonitrile (nitrilase gene). Przibilla, et al., (1991) Plant Cell3:169, describe the transformation of Chlamydomonas with plasmidsencoding mutant psbA genes. Nucleotide sequences for nitrilase genes aredisclosed in U.S. Pat. No. 4,810,648 to Stalker and DNA moleculescontaining these genes are available under ATCC® Accession Numbers53435, 67441 and 67442. Cloning and expression of DNA coding for aglutathione S-transferase is described by Hayes, et al., (1992) Biochem.J. 285:173.

(D) A polynucleotide encoding a protein for resistance to Acetohydroxyacid synthase, which has been found to make plants that express thisenzyme resistant to multiple types of herbicides, has been introducedinto a variety of plants (see, e.g., Hattori, et al., (1995) Mol GenGenet. 246:419). Other genes that confer resistance to herbicidesinclude: a gene encoding a chimeric protein of rat cytochrome P4507A1and yeast NADPH-cytochrome P450 oxidoreductase (Shiota, et al., (1994)Plant Physiol 106:17), genes for glutathione reductase and superoxidedismutase (Aono, et al., (1995) Plant Cell Physiol 36:1687) and genesfor various phosphotransferases (Datta, et al., (1992) Plant Mol Biol20:619).

(E) A polynucleotide encoding resistance to a herbicide targetingProtoporphyrinogen oxidase (protox) which is necessary for theproduction of chlorophyll. The protox enzyme serves as the target for avariety of herbicidal compounds. These herbicides also inhibit growth ofall the different species of plants present, causing their totaldestruction. The development of plants containing altered protoxactivity which are resistant to these herbicides are described in U.S.Pat. Nos. 6,288,306; 6,282,83 and 5,767,373 and InternationalPublication WO 2001/12825.

(F) The aad-1 gene (originally from Sphingobium herbicidovorans) encodesthe aryloxyalkanoate dioxygenase (AAD-1) protein. The trait conferstolerance to 2,4-dichlorophenoxyacetic acid and aryloxyphenoxypropionate(commonly referred to as “fop” herbicides such as quizalofop)herbicides. The aad-1 gene, itself, for herbicide tolerance in plantswas first disclosed in WO 2005/107437 (see also, US 2009/0093366). Theaad-12 gene, derived from Delftia acidovorans, which encodes thearyloxyalkanoate dioxygenase (AAD-12) protein that confers tolerance to2,4-dichlorophenoxyacetic acid and pyridyloxyacetate herbicides bydeactivating several herbicides with an aryloxyalkanoate moiety,including phenoxy auxin (e.g., 2,4-D, MCPA), as well as pyridyloxyauxins (e.g., fluroxypyr, triclopyr).

(G) A polynucleotide encoding a herbicide resistant dicambamonooxygenase disclosed in US Patent Application Publication2003/0135879 for imparting dicamba tolerance;

(H) A polynucleotide molecule encoding bromoxynil nitrilase (Bxn)disclosed in U.S. Pat. No. 4,810,648 for imparting bromoxynil tolerance;

(I) A polynucleotide molecule encoding phytoene (crtl) described inMisawa, et al., (1993) Plant J. 4:833-840 and in Misawa, et al., (1994)Plant J. 6:481-489 for norflurazon tolerance.

3. Transgenes that confer or contribute to an altered graincharacteristic;

4. Genes that control male sterility;

5. Genes that create a site for site specific DNA integration;

6. Genes that affect abiotic stress resistance;

7. Genes that confer increased yield; and/or

8. Genes that confer plant digestibility.

9. Gene silencing. In some embodiments, the stacked trait may be in theform of silencing of one or more polynucleotides of interest resultingin suppression of one or more target pest polypeptides. In someembodiments, the silencing is achieved using a suppression DNAconstruct.

In some embodiments, one or more polynucleotide encoding the IRDIG35563polypeptides or fragments or variants thereof may be stacked with one ormore polynucleotides encoding one or more polypeptides havinginsecticidal activity or agronomic traits as set forth supra andoptionally may further include one or more polynucleotides providing forgene silencing of one or more target polynucleotides as discussed infra.

“Suppression DNA construct” is a recombinant DNA construct which whentransformed or stably integrated into the genome of the plant, resultsin “silencing” of a target gene in the plant. The target gene may beendogenous or transgenic to the plant. “Silencing,” as used herein withrespect to the target gene, refers generally to the suppression oflevels of mRNA or protein/enzyme expressed by the target gene, and/orthe level of the enzyme activity or protein functionality. The term“suppression” includes lower, reduce, decline, decrease, inhibit,eliminate and prevent. “Silencing” or “gene silencing” does not specifymechanism and is inclusive, and not limited to, anti-sense,cosuppression, viral-suppression, hairpin suppression, stem-loopsuppression, RNAi-based approaches and small RNA-based approaches.

Some embodiments relate to down-regulation of expression of target genesin insect pest species by interfering ribonucleic acid (RNA) molecules.PCT Publication WO 2007/074405 describes methods of inhibitingexpression of target genes in invertebrate pests including Coloradopotato beetle. PCT Publication WO 2005/110068 describes methods ofinhibiting expression of target genes in invertebrate pests includingWestern corn rootworm to control insect infestation. Furthermore, PCTPublication WO 2009/091864 describes compositions and methods for thesuppression of target genes from insect pest species including pestsfrom the Lygus genus. Nucleic acid molecules including RNAi fortargeting the vacuolar ATPase H subunit, useful for controlling acoleopteran pest population and infestation as described in US PatentApplication Publication 2012/0198586. PCT Publication WO 2012/055982describes ribonucleic acid (RNA or double stranded RNA) that inhibits ordown regulates the expression of a target gene that encodes: an insectribosomal protein such as the ribosomal protein L19, the ribosomalprotein L40 or the ribosomal protein S27A; an insect proteasome subunitsuch as the Rpn6 protein, the Pros 25, the Rpn2 protein, the proteasomebeta 1 subunit protein or the Pros beta 2 protein; an insect β-coatomerof the COPI vesicle, the γ-coatomer of the COPI vesicle, the β′-coatomerprotein or the ζ-coatomer of the COPI vesicle; an insect Tetraspanine 2A protein which is a putative transmembrane domain protein; an insectprotein belonging to the actin family such as Actin 5C; an insectubiquitin-5E protein; an insect Sec23 protein which is a GTPaseactivator involved in intracellular protein transport; an insectcrinkled protein which is an unconventional myosin which is involved inmotor activity; an insect crooked neck protein which is involved in theregulation of nuclear alternative mRNA splicing; an insect vacuolarH+-ATPase G-subunit protein and an insect Tbp-1 such as Tat-bindingprotein. PCT publication WO 2007/035650 describes ribonucleic acid (RNAor double stranded RNA) that inhibits or down regulates the expressionof a target gene that encodes Snf7. US Patent Application publication2011/0054007 describes polynucleotide silencing elements targetingRPS10. US Patent Application publications 2014/0275208 andUS2015/0257389 describe polynucleotide silencing elements targetingRyanR and PAT3. PCT Patent Application publication WO2016/138106describes polynucleotide silencing elements targeting coatomer alpha orgamma. US Patent Application Publications 2012/029750, US 20120297501,and 2012/0322660 describe interfering ribonucleic acids (RNA or doublestranded RNA) that functions upon uptake by an insect pest species todown-regulate expression of a target gene in said insect pest, whereinthe RNA comprises at least one silencing element wherein the silencingelement is a region of double-stranded RNA comprising annealedcomplementary strands, one strand of which comprises or consists of asequence of nucleotides which is at least partially complementary to atarget nucleotide sequence within the target gene. US Patent ApplicationPublication 2012/0164205 describe potential targets for interferingdouble stranded ribonucleic acids for inhibiting invertebrate pestsincluding: a Chd3 Homologous Sequence, a Beta-Tubulin HomologousSequence, a 40 kDa V-ATPase Homologous Sequence, a EF1α HomologousSequence, a 26S Proteosome Subunit p28 Homologous Sequence, a JuvenileHormone Epoxide Hydrolase Homologous Sequence, a Swelling DependentChloride Channel Protein Homologous Sequence, a Glucose-6-Phosphate1-Dehydrogenase Protein Homologous Sequence, an Act42A ProteinHomologous Sequence, a ADP-Ribosylation Factor 1 Homologous Sequence, aTranscription Factor IIB Protein Homologous Sequence, a ChitinaseHomologous Sequences, a Ubiquitin Conjugating Enzyme HomologousSequence, a Glyceraldehyde-3-Phosphate Dehydrogenase HomologousSequence, an Ubiquitin B Homologous Sequence, a Juvenile HormoneEsterase Homolog, and an Alpha Tubulin Homologous Sequence.

Use in Pesticidal Control. General methods for employing strainscomprising a nucleic acid sequence of the embodiments or a variantthereof, in pesticide control or in engineering other organisms aspesticidal agents are known in the art.

Microorganism hosts that are known to occupy the “phytosphere”(phylloplane, phyllosphere, rhizosphere, and/or rhizoplana) of one ormore crops of interest may be selected. These microorganisms areselected to be capable of successfully competing in the particularenvironment with the wild-type microorganisms, provide for stablemaintenance and expression of the gene expressing the IRDIG35563polypeptide and desirably provide for improved protection of thepesticide from environmental degradation and inactivation.

Alternatively, the IRDIG35563 polypeptide is produced by introducing aheterologous gene into a cellular host. Expression of the heterologousgene results, directly or indirectly, in the intracellular productionand maintenance of the pesticide. These cells are then treated underconditions that prolong the activity of the toxin produced in the cellwhen the cell is applied to the environment of target pest(s). Theresulting product retains the toxicity of the toxin. These naturallyencapsulated IRDIG35563 polypeptides may then be formulated inaccordance with conventional techniques for application to theenvironment hosting a target pest, e.g., soil, water, and foliage ofplants. See, for example EPA 0192319, and the references cited therein.

Spray-on applications are another example and are also known in the art.The subject proteins can be appropriately formulated for the desired enduse, and then sprayed (or otherwise applied) onto the plant or aroundthe plant or to the vicinity of the plant to be protected before aninfestation is discovered, after target insects are discovered, bothbefore and after, and the like. Bait granules, for example, can also beused and are known in the art.

Pesticidal compositions. In some embodiments, the active ingredients canbe applied in the form of compositions and can be applied to the croparea or plant to be treated, simultaneously or in succession, with othercompounds. These compounds can be fertilizers, weed killers,cryoprotectants, surfactants, detergents, pesticidal soaps, dormantoils, polymers, and/or time-release or biodegradable carrierformulations that permit long-term dosing of a target area following asingle application of the formulation. They can also be selectiveherbicides, chemical insecticides, virucides, microbicides, amoebicides,pesticides, fungicides, bacteriocides, nematocides, molluscicides ormixtures of several of these preparations, if desired, together withfurther agriculturally acceptable carriers, surfactants orapplication-promoting adjuvants can be employed in the formulation.Suitable carriers and adjuvants can be solid or liquid and correspond tothe substances ordinarily employed in formulation technology, e.g.,natural or regenerated mineral substances, solvents, dispersants,wetting agents, tackifiers, binders or fertilizers. Likewise, theformulations may be prepared into edible “baits” or fashioned into pest“traps” to permit feeding or ingestion by a target pest of thepesticidal formulation.

Methods of applying an active ingredient or an agrochemical compositionthat contains at least one of the IRDIG35563 polypeptide produced by thebacterial strains include leaf application, seed coating and soilapplication. The number of applications and the rate of applicationdepend on the intensity of infestation by the corresponding pest.

The composition may be formulated as a powder, dust, pellet, granule,spray, emulsion, colloid, solution or such like, and may be prepared bysuch conventional means as desiccation, lyophilization, homogenation,extraction, filtration, centrifugation, sedimentation or concentrationof a culture of cells comprising the polypeptide. In all suchcompositions that contain at least one such pesticidal polypeptide, thepolypeptide may be present in a concentration of from about 1% to about99% by weight.

Lepidopteran, Dipteran, Heteropteran, nematode, Hemiptera or Coleopteranpests may be killed or reduced in numbers in each area by the methods ofthe disclosure or may be prophylactically applied to an environmentalarea to prevent infestation by a susceptible pest. Preferably the pestingests or is contacted with, a pesticidally-effective amount of thepolypeptide. “Pesticidally-effective amount” as used herein refers to anamount of the pesticide that can bring about death to at least one pestor to noticeably reduce pest growth, feeding or normal physiologicaldevelopment. This amount will vary depending on such factors as, forexample, the specific target pests to be controlled, the specificenvironment, location, plant, crop or agricultural site to be treated,the environmental conditions and the method, rate, concentration,stability, and quantity of application of the pesticidally-effectivepolypeptide composition. The formulations may also vary with respect toclimatic conditions, environmental considerations, and/or frequency ofapplication and/or severity of pest infestation.

The pesticide compositions described may be made by formulating thebacterial cell, Crystal and/or spore suspension or isolated proteincomponent with the desired agriculturally-acceptable carrier. Thecompositions may be formulated prior to administration in an appropriatemeans such as lyophilized, freeze-dried, desiccated or in an aqueouscarrier, medium or suitable diluent, such as saline or another buffer.The formulated compositions may be in the form of a dust or granularmaterial or a suspension in oil (vegetable or mineral) or water oroil/water emulsions or as a wettable powder or in combination with anyother carrier material suitable for agricultural application. Suitableagricultural carriers can be solid or liquid. The term“agriculturally-acceptable carrier” covers all adjuvants, inertcomponents, dispersants, surfactants, tackifiers, binders, etc. that areordinarily used in pesticide formulation technology; these are wellknown to those skilled in pesticide formulation. The formulations may bemixed with one or more solid or liquid adjuvants and prepared by variousmeans, e.g., by homogeneously mixing, blending and/or grinding thepesticidal composition with suitable adjuvants using conventionalformulation techniques. Suitable formulations and application methodsare described in U.S. Pat. No. 6,468,523, herein incorporated byreference. The plants can also be treated with one or more chemicalcompositions, including one or more herbicide, insecticides orfungicides.

Exemplary chemical compositions include: Fruits/Vegetables Herbicides:Atrazine, Bromacil, Diuron, Glyphosate, Linuron, Metribuzin, Simazine,Trifluralin, Fluazifop, Glufosinate, Halo sulfuron Gowan, Paraquat,Propyzamide, Sethoxydim, Butafenacil, Halosulfuron, Indaziflam;Fruits/Vegetables Insecticides: Aldicarb, Bacillus thuringiensis,Carbaryl, Carbofuran, Chlorpyrifos, Cypermethrin, Deltamethrin,Diazinon, Malathion, Abamectin, Cyfluthrin/beta-cyfluthrin,Esfenvalerate, Lambda-cyhalothrin, Acequinocyl, Bifenazate,Methoxyfenozide, Novaluron, Chromafenozide, Thiacloprid, Dinotefuran,FluaCrypyrim, Tolfenpyrad, Clothianidin, Spirodiclofen,Gamma-cyhalothrin, Spiromesifen, Spinosad, Rynaxypyr, Cyazypyr,Spinoteram, Triflumuron, Spirotetramat, Imidacloprid, Flubendiamide,Thiodicarb, Metaflumizone, Sulfoxaflor, Cyflumetofen, Cyanopyrafen,Imidacloprid, Clothianidin, Thiamethoxam, Spinotoram, Thiodicarb,Flonicamid, Methiocarb, Emamectin-benzoate, Indoxacarb, Forthiazate,Fenamiphos, Cadusaphos, Pyriproxifen, Fenbutatin-oxid, Hexthiazox,Methomyl,4-[[(6-Chlorpyridin-3-yl)methyl](2,2-difluorethyl)amino]furan-2(5H)-on;Fruits/Vegetables Fungicides: Carbendazim, Chlorothalonil, EBDCs,Sulphur, Thiophanate-methyl, Azoxystrobin, Cymoxanil, Fluazinam,Fosetyl, Iprodione, Kresoxim-methyl, Metalaxyl/mefenoxam,Trifloxystrobin, Ethaboxam, Iprovalicarb, Trifloxystrobin, Fenhexamid,Oxpoconazole fumarate, Cyazofamid, Fenamidone, Zoxamide, Picoxystrobin,Pyraclostrobin, Cyflufenamid, Boscalid; Cereals Herbicides: Isoproturon,Bromoxynil, Ioxynil, Phenoxies, Chlorsulfuron, Clodinafop, Diclofop,Diflufenican, Fenoxaprop, Florasulam, Fluoroxypyr, Metsulfuron,Triasulfuron, Flucarbazone, Iodosulfuron, Propoxycarbazone, Picolinafen,Mesosulfuron, Beflubutamid, Pinoxaden, Amidosulfuron, ThifensulfuronMethyl, Tribenuron, Flupyrsulfuron, Sulfosulfuron, Pyrasulfotole,Pyroxsulam, Flufenacet, Tralkoxydim, Pyroxasulfon; Cereals Fungicides:Carbendazim, Chlorothalonil, Azoxystrobin, Cyproconazole, Cyprodinil,Fenpropimorph, Epoxiconazole, Kresoxim-methyl, Quinoxyfen, Tebuconazole,Trifloxystrobin, Simeconazole, Picoxystrobin, Pyraclostrobin,Dimoxystrobin, Prothioconazole, Fluoxastrobin; Cereals Insecticides:Dimethoate, Lambda-cyhalthrin, Deltamethrin, alpha-Cypermethrin,β-cyfluthrin, Bifenthrin, Imidacloprid, Clothianidin, Thiamethoxam,Thiacloprid, Acetamiprid, Dinetofuran, Clorphyriphos, Metamidophos,Oxidemethon-methyl, Pirimicarb, Methiocarb; Maize Herbicides: Atrazine,Alachlor, Bromoxynil, Acetochlor, Dicamba, Clopyralid, (S-)Dimethenamid, Glufosinate, Glyphosate, Isoxaflutole, (S-)Metolachlor,Mesotrione, Nicosulfuron, Primisulfuron, Rimsulfuron, Sulcotrione,Foramsulfuron, Topramezone, Tembotrione, Saflufenacil, Thiencarbazone,Flufenacet, Pyroxasulfon; Maize Insecticides: Carbofuran, Chlorpyrifos,Bifenthrin, Fipronil, Imidacloprid, Lambda-Cyhalothrin, Tefluthrin,Terbufos, Thiamethoxam, Clothianidin, Spiromesifen, Flubendiamide,Triflumuron, Rynaxypyr, Deltamethrin, Thiodicarb, β-Cyfluthrin,Cypermethrin, Bifenthrin, Lufenuron, Triflumoron, Tefluthrin,Tebupirimphos, Ethiprole, Cyazypyr, Thiacloprid, Acetamiprid,Dinetofuran, Avermectin, Methiocarb, Spirodiclofen, Spirotetramat; MaizeFungicides: Fenitropan, Thiram, Prothioconazole, Tebuconazole,Trifloxystrobin; Rice Herbicides: Butachlor, Propanil, Azimsulfuron,Bensulfuron, Cyhalofop, Daimuron, Fentrazamide, Imazosulfuron,Mefenacet, Oxaziclomefone, Pyrazosulfuron, Pyributicarb, Quinclorac,Thiobencarb, Indanofan, Flufenacet, Fentrazamide, Halosulfuron,Oxaziclomefone, Benzobicyclon, Pyriftalid, Penoxsulam, Bispyribac,Oxadiargyl, Ethoxysulfuron, Pretilachlor, Mesotrione, Tefuryltrione,Oxadiazone, Fenoxaprop, Pyrimisulfan; Rice Insecticides: Diazinon,Fenitrothion, Fenobucarb, Monocrotophos, Benfuracarb, Buprofezin,Dinotefuran, Fipronil, Imidacloprid, Isoprocarb, Thiacloprid,Chromafenozide, Thiacloprid, Dinotefuran, Clothianidin, Ethiprole,Flubendiamide, Rynaxypyr, Deltamethrin, Acetamiprid, Thiamethoxam,Cyazypyr, Spinosad, Spinotoram, Emamectin-Benzoate, Cypermethrin,Chlorpyriphos, Cartap, Methamidophos, Etofenprox, Triazophos,4-[[(6-Chlorpyridin-3-yl)methyl](2,2-difluorethyl)amino]furan-2(5H)-on,Carbofuran, Benfuracarb; Rice Fungicides: Thiophanate-methyl,Azoxystrobin, Carpropamid, Edifenphos, Ferimzone, Iprobenfos,Isoprothiolane, Pencycuron, Probenazole, Pyroquilon, Tricyclazole,Trifloxystrobin, Diclocymet, Fenoxanil, Simeconazole, Tiadinil; CottonHerbicides: Diuron, Fluometuron, MSMA, Oxyfluorfen, Prometryn,Trifluralin, Carfentrazone, Clethodim, Fluazifop-butyl, Glyphosate,Norflurazon, Pendimethalin, Pyrithiobac-sodium, Trifloxysulfuron,Tepraloxydim, Glufosinate, Flumioxazin, Thidiazuron; CottonInsecticides: Acephate, Aldicarb, Chlorpyrifos, Cypermethrin,Deltamethrin, Malathion, Monocrotophos, Abamectin, Acetamiprid,Emamectin Benzoate, Imidacloprid, Indoxacarb, Lambda-Cyhalothrin,Spinosad, Thiodicarb, Gamma-Cyhalothrin, Spiromesifen, Pyridalyl,Flonicamid, Flubendiamide, Triflumuron, Rynaxypyr, Beta-Cyfluthrin,Spirotetramat, Clothianidin, Thiamethoxam, Thiacloprid, Dinetofuran,Flubendiamide, Cyazypyr, Spinosad, Spinotoram, gamma Cyhalothrin,4-[[(6-Chlorpyridin-3-yl)methyl](2,2-difluorethyl)amino]furan-2(5H)-on,Thiodicarb, Avermectin, Flonicamid, Pyridalyl, Spiromesifen,Sulfoxaflor, Profenophos, Thriazophos, Endosulfan; Cotton Fungicides:Etridiazole, Metalaxyl, Quintozene; Soybean Herbicides: Alachlor,Bentazone, Trifluralin, Chlorimuron-Ethyl, Cloransulam-Methyl,Fenoxaprop, Fomesafen, Fluazifop, Glyphosate, Imazamox, Imazaquin,Imazethapyr, (S-)Metolachlor, Metribuzin, Pendimethalin, Tepraloxydim,Glufosinate; Soybean Insecticides: Lambda-cyhalothrin, Methomyl,Parathion, Thiocarb, Imidacloprid, Clothianidin, Thiamethoxam,Thiacloprid, Acetamiprid, Dinetofuran, Flubendiamide, Rynaxypyr,Cyazypyr, Spinosad, Spinotoram, Emamectin-Benzoate, Fipronil, Ethiprole,Deltamethrin, β-Cyfluthrin, gamma and lambda Cyhalothrin,4-[[(6-Chlorpyridin-3-yl)methyl](2,2-difluorethyl)amino]furan-2(5H)-on,Spirotetramat, Spinodiclofen, Triflumuron, Flonicamid, Thiodicarb,beta-Cyfluthrin; Soybean Fungicides: Azoxystrobin, Cyproconazole,Epoxiconazole, Flutriafol, Pyraclostrobin, Tebuconazole,Trifloxystrobin, Prothioconazole, Tetraconazole; Sugarbeet Herbicides:Chloridazon, Desmedipham, Ethofumesate, Phenmedipham, Triallate,Clopyralid, Fluazifop, Lenacil, Metamitron, Quinmerac, Cycloxydim,Triflusulfuron, Tepraloxydim, Quizalofop; Sugarbeet Insecticides:Imidacloprid, Clothianidin, Thiamethoxam, Thiacloprid, Acetamiprid,Dinetofuran, Deltamethrin, β-Cyfluthrin, gamma/lambda Cyhalothrin,4-[[(6-Chlorpyridin-3-yl)methyl](2,2-difluorethyl)amino]furan-2(5H)-on,Tefluthrin, Rynaxypyr, Cyaxypyr, Fipronil, Carbofuran; CanolaHerbicides: Clopyralid, Diclofop, Fluazifop, Glufosinate, Glyphosate,Metazachlor, Trifluralin Ethametsulfuron, Quinmerac, Quizalofop,Clethodim, Tepraloxydim; Canola Fungicides: Azoxystrobin, Carbendazim,Fludioxonil, Iprodione, Prochloraz, Vinclozolin; Canola Insecticides:Carbofuran organophosphates, Pyrethroids, Thiacloprid, Deltamethrin,Imidacloprid, Clothianidin, Thiamethoxam, Acetamiprid, Dinetofuran,O-Cyfluthrin, gamma and lambda Cyhalothrin, tau-Fluvaleriate, Ethiprole,Spinosad, Spinotoram, Flubendiamide, Rynaxypyr, Cyazypyr,4-[[(6-Chlorpyridin-3-yl)methyl](2,2-difluorethyl)amino]furan-2(5H)-on.

In some embodiments, the herbicide is Atrazine, Bromacil, Diuron,Chlorsulfuron, Metsulfuron, Thifensulfuron Methyl, Tribenuron,Acetochlor, Dicamba, Isoxaflutole, Nicosulfuron, Rimsulfuron,Pyrithiobac-sodium, Flumioxazin, Chlorimuron-Ethyl, Metribuzin,Quizalofop, S-metolachlor, Hexazinne or combinations thereof.

In some embodiments, the insecticide is Esfenvalerate,Chlorantraniliprole, Methomyl, Indoxacarb, Oxamyl or combinationsthereof.

Pesticidal and insecticidal activity. “Pest” includes but is not limitedto, insects, fungi, bacteria, nematodes, mites, ticks and the like.Insect pests include insects selected from the orders Coleoptera,Diptera, Hymenoptera, Lepidoptera, Mallophaga, Homoptera, HemipteraOrthroptera, Thysanoptera, Dermaptera, Isoptera, Anoplura, Siphonaptera,Trichoptera, etc., particularly Lepidoptera and Coleoptera.

Compounds of the embodiments display activity against insect pests,which may include economically important agronomic, forest, greenhouse,nursery ornamentals, food and fiber, public and animal health, domesticand commercial structure, household and stored product pests.

Larvae of the order Lepidoptera include, but are not limited to,armyworms, cutworms, loopers and heliothines in the family NoctuidaeSpodoptera frugiperda JE Smith (fall armyworm); S. exigua Hübner (beetarmyworm); S. litura Fabricius (tobacco cutworm, cluster caterpillar);Mamestra configurata Walker (bertha armyworm); M. brassicae Linnaeus(cabbage moth); Agrotis ipsilon Hufnagel (black cutworm); A. orthogoniaMorrison (western cutworm); A. subterranea Fabricius (granulatecutworm); Alabama argillacea Hübner (cotton leaf worm); Trichoplusia niHübner (cabbage looper); Pseudoplusia includens Walker (soybean looper);Anticarsia gemmatalis Hübner (velvetbean caterpillar); Hypena scabraFabricius (green cloverworm); Heliothis virescens Fabricius (tobaccobudworm); Pseudaletia unipuncta Haworth (armyworm); Athetis mindaraBarnes and Mcdunnough (rough skinned cutworm); Euxoa messoria Harris(darksided cutworm); Earias insulana Boisduval (spiny bollworm); E.vittella Fabricius (spotted bollworm); Helicoverpa armigera Hübner(American bollworm); H. zea Boddie (corn earworm or cotton bollworm);Melanchrapicta Harris (zebra caterpillar); Egira (Xylomyges) curialisGrote (citrus cutworm); borers, casebearers, webworms, coneworms, andskeletonizers from the family Pyralidae Ostrinia nubilalis Hübner(European corn borer); Amyelois transitella Walker (naval orangeworm);Anagasta kuehniella Zeller (Mediterranean flour moth); Cadra cautellaWalker (almond moth); Chilo suppressalis Walker (rice stem borer); C.partellus, (sorghum borer); Corcyra cephalonica Stainton (rice moth);Crambus caliginosellus Clemens (corn root webworm); C. teterrellusZincken (bluegrass webworm); Cnaphalocrocis medinalis Guenée (rice leafroller); Desmia funeralis Hübner (grape leaffolder); Diaphania hyalinataLinnaeus (melon worm); D. nitidalis Stoll (pickleworm); Diatraeagrandiosella Dyar (southwestern corn borer), D. saccharalis Fabricius(surgarcane borer); Eoreuma loftini Dyar (Mexican rice borer); Ephestiaelutella Hübner (tobacco (cacao) moth); Galleria mellonella Linnaeus(greater wax moth); Herpetogramma licarsisalis Walker (sod webworm);Homoeosoma electellum Hulst (sunflower moth); Elasmopalpus lignosellusZeller (lesser cornstalk borer); Achroia grisella Fabricius (lesser waxmoth); Loxostege sticticalis Linnaeus (beet webworm); Orthaga thyrisalisWalker (tea tree web moth); Maruca testulalis Geyer (bean pod borer);Plodia interpunctella Hübner (Indian meal moth); Scirpophaga incertulasWalker (yellow stem borer); Udea rubigalis Guenée (celery leaftier); andleafrollers, budworms, seed worms and fruit worms in the familyTortricidae Acleris gloverana Walsingham (Western blackheaded budworm);A. variana Fernald (Eastern blackheaded budworm); Archips argyrospilaWalker (fruit tree leaf roller); A. rosana Linnaeus (European leafroller); and other Archips species, Adoxophyes orana Fischer vonRbsslerstamm (summer fruit tortrix moth); Cochylis hospes Walsingham(banded sunflower moth); Cydia latiferreana Walsingham (filbertworm); C.pomonella Linnaeus (coding moth); Platynota flavedana Clemens(variegated leafroller); P. stultana Walsingham (omnivorous leafroller);Lobesia botrana Denis & Schiffermuller (European grape vine moth);Spilonota ocellana Denis & Schiffermuller (eyespotted bud moth);Endopiza viteana Clemens (grape berry moth); Eupoecilia ambiguellaHübner (vine moth); Bonagota salubricola Meyrick (Brazilian appleleafroller); Grapholita molesta Busck (oriental fruit moth); Suleimahelianthana Riley (sunflower bud moth); Argyrotaenia spp.; andChoristoneura spp.

Selected other agronomic pests in the order Lepidoptera include, but arenot limited to, Alsophila pometaria Harris (fall cankerworm); Anarsialineatella Zeller (peach twig borer); Anisota senatoria J. E. Smith(orange striped oakworm); Antheraea pernyi Guerin-Meneville (Chinese OakTussah Moth); Bombyx mori Linnaeus (Silkworm); Bucculatrix thurberiellaBusck (cotton leaf perforator); Colias eurytheme Boisduval (alfalfacaterpillar); Datana integerrima Grote & Robinson (walnut caterpillar);Dendrolimus sibiricus Tschetwerikov (Siberian silk moth), Ennomossubsignaria Hübner (elm spanworm); Erannis tiliaria Harris (lindenlooper); Euproctis chrysorrhoea Linnaeus (browntail moth); Harrisinaamericana Guerin-Meneville (grapeleaf skeletonizer); Hemileuca oliviaeCockrell (range caterpillar); Hyphantria cunea Drury (fall webworm);Keiferia lycopersicella Walsingham (tomato pinworm); Lambdinafiscellaria fscellaria Hulst (Eastern hemlock looper); L. fiscellarialugubrosa Hulst (Western hemlock looper); Leucoma salicis Linnaeus(satin moth); Lymantria dispar Linnaeus (gypsy moth); Manducaquinquemaculata Haworth (five spotted hawk moth, tomato hornworm); M.sexta Haworth (tomato hornworm, tobacco hornworm); Operophtera brumataLinnaeus (winter moth); Paleacrita vernata Peck (spring cankerworm);Papilio cresphontes Cramer (giant swallowtail orange dog); Phryganidiacalifornica Packard (California oakworm); Phyllocnistis citrellaStainton (citrus leafminer); Phyllonorycter blancardella Fabricius(spotted tentiform leafminer); Pieris brassicae Linnaeus (large whitebutterfly); P. rapae Linnaeus (small white butterfly); P. napi Linnaeus(green veined white butterfly); Platyptilia carduidactyla Riley(artichoke plume moth); Plutella xylostella Linnaeus (diamondback moth);Pectinophora gossypiella Saunders (pink bollworm); Pontia protodiceBoisduval and Leconte (Southern cabbageworm); Sabulodes aegrotata Guenée(omnivorous looper); Schizura concinna J. E. Smith (red humpedcaterpillar); Sitotroga cerealella Olivier (Angoumois grain moth);Thaumetopoea pityocampa Schiffermuller (pine processionary caterpillar);Tineola bisselliella Hummel (webbing clothesmoth); Tuta absoluta Meyrick(tomato leafminer); Yponomeuta padella Linnaeus (ermine moth); Heliothissubflexa Guenée; Malacosoma spp. and Orgyia spp.

Of interest are larvae and adults of the order Coleoptera includingweevils from the families Anthribidae, Bruchidae and Curculionidae(including, but not limited to: Anthonomus grandis Boheman (bollweevil); Lissorhoptrus oryzophilus Kuschel (rice water weevil);Sitophilus granarius Linnaeus (granary weevil); S. oryzae Linnaeus (riceweevil); Hypera punctata Fabricius (clover leaf weevil);Cylindrocopturus adspersus LeConte (sunflower stem weevil); Smicronyxfulvus LeConte (red sunflower seed weevil); S. sordidus LeConte (graysunflower seed weevil); Sphenophorus maidis Chittenden (maize billbug));flea beetles, cucumber beetles, rootworms, leaf beetles, potato beetlesand leafminers in the family Chrysomelidae (including, but not limitedto: Leptinotarsa decemlineata Say (Colorado potato beetle); Diabroticavirgifera virgifera LeConte (western corn rootworm); D. barberi Smithand Lawrence (northern corn rootworm); D. undecimpunctata howardi Barber(southern corn rootworm); Chaetocnema pulicaria Melsheimer (corn fleabeetle); Phyllotreta cruciferae Goeze (Crucifer flea beetle);Phyllotreta striolata (stripped flea beetle); Colaspis brunnea Fabricius(grape colaspis); Oulema melanopus Linnaeus (cereal leaf beetle);Zygogramma exclamationis Fabricius (sunflower beetle)); beetles from thefamily Coccinellidae (including, but not limited to: Epilachnavarivestis Mulsant (Mexican bean beetle)); chafers and other beetlesfrom the family Scarabaeidae (including, but not limited to: Popilliajaponica Newman (Japanese beetle); Cyclocephala borealis Arrow (northernmasked chafer, white grub); C. immaculata Olivier (southern maskedchafer, white grub); Rhizotrogus majalis Razoumowsky (European chafer);Phyllophaga crinita Burmeister (white grub); Ligyrus gibbosus De Geer(carrot beetle)); carpet beetles from the family Dermestidae; wirewormsfrom the family Elateridae, Eleodes spp., Melanotus spp.; Conoderusspp.; Limonius spp.; Agriotes spp.; Ctenicera spp.; Aeolus spp.; barkbeetles from the family Scolytidae and beetles from the familyTenebrionidae.

Adults and immatures of the order Diptera are of interest, includingleafminers Agromyza parvicornis Loew (corn blotch leafminer); midges(including, but not limited to: Contarinia sorghicola Coquillett(sorghum midge); Mayetiola destructor Say (Hessian fly); Sitodiplosismosellana Gehin (wheat midge); Neolasioptera murtfeldtiana Felt,(sunflower seed midge)); fruit flies (Tephritidae), Oscinella fritLinnaeus (fruit flies); maggots (including, but not limited to: Deliaplatura Meigen (seedcorn maggot); D. coarctata Fallen (wheat bulb fly)and other Delia spp., Meromyza americana Fitch (wheat stem maggot);Musca domestica Linnaeus (house flies); Fannia canicularis Linnaeus, F.femoralis Stein (lesser house flies); Stomoxys calcitrans Linnaeus(stable flies)); face flies, horn flies, blow flies, Chrysomya spp.;Phormia spp. and other muscoid fly pests, horse flies Tabanus spp.; botflies Gastrophilus spp.; Oestrus spp.; cattle grubs Hypoderma spp.; deerflies Chrysops spp.; Melophagus ovinus Linnaeus (keds) and otherBrachycera, mosquitoes Aedes spp.; Anopheles spp.; Culex spp.; blackflies Prosimulium spp.; Simulium spp.; biting midges, sand flies,sciarids, and other Nematocera.

Included as insects of interest are adults and nymphs of the ordersHemiptera and Homoptera such as, but not limited to, adelgids from thefamily Adelgidae, plant bugs from the family Miridae, cicadas from thefamily Cicadidae, leafhoppers, Empoasca spp.; from the familyCicadellidae, planthoppers from the families Cixiidae, Flatidae,Fulgoroidea, Issidae and Delphacidae, treehoppers from the familyMembracidae, psyllids from the family Psyllidae, whiteflies from thefamily Aleyrodidae, aphids from the family Aphididae, phylloxera fromthe family Phylloxeridae, mealybugs from the family Pseudococcidae,scales from the families Asterolecanidae, Coccidae, Dactylopiidae,Diaspididae, Eriococcidae Ortheziidae, Phoenicococcidae andMargarodidae, lace bugs from the family Tingidae, stink bugs from thefamily Pentatomidae, cinch bugs, Blissus spp.; and other seed bugs fromthe family Lygaeidae, spittlebugs from the family Cercopidae squash bugsfrom the family Coreidae and red bugs and cotton stainers from thefamily Pyrrhocoridae.

Agronomically important members from the order Homoptera furtherinclude, but are not limited to: Acyrthisiphon pisum Harris (pea aphid);Aphis craccivora Koch (cowpea aphid); A. fabae Scopoli (black beanaphid); A. gossypii Glover (cotton aphid, melon aphid); A. maidiradicisForbes (corn root aphid); A. pomi De Geer (apple aphid); A. spiraecolaPatch (spirea aphid); Aulacorthum solani Kaltenbach (foxglove aphid);Chaetosiphon fragaefolii Cockerell (strawberry aphid); Diuraphis noxiaKurdjumov/Mordvilko (Russian wheat aphid); Dysaphis plantagineaPaaserini (rosy apple aphid); Eriosoma lanigerum Hausmann (woolly appleaphid); Brevicoryne brassicae Linnaeus (cabbage aphid); Hyalopteruspruni Geoffroy (mealy plum aphid); Lipaphis erysimi Kaltenbach (turnipaphid); Metopolophium dirrhodum Walker (cereal aphid); Macrosiphumeuphorbiae Thomas (potato aphid); Myzus persicae Sulzer (peach-potatoaphid, green peach aphid); Nasonovia ribisnigri Mosley (lettuce aphid);Pemphigus spp. (root aphids and gall aphids); Rhopalosiphum maidis Fitch(corn leaf aphid); R. padi Linnaeus (bird cherry-oat aphid); Schizaphisgraminum Rondani (greenbug); Sipha flava Forbes (yellow sugarcaneaphid); Sitobion avenae Fabricius (English grain aphid); Therioaphismaculata Buckton (spotted alfalfa aphid); Toxoptera aurantii Boyer deFonscolombe (black citrus aphid) and T. citricida Kirkaldy (brown citrusaphid); Adelges spp. (adelgids); Phylloxera devastatrix Pergande (pecanphylloxera); Bemisia tabaci Gennadius (tobacco whitefly, sweetpotatowhitefly); B. argentifoli Bellows & Perring (silverleaf whitefly);Dialeurodes citri Ashmead (citrus whitefly); Trialeurodes abutiloneus(bandedwinged whitefly) and T. vaporariorum Westwood (greenhousewhitefly); Empoasca fabae Harris (potato leafhopper); Laodelphaxstriatellus Fallen (smaller brown planthopper); Macrolestesquadrilineatus Forbes (aster leafhopper); Nephotettix cinticeps Uhler(green leafhopper); N. nigropictus Stil (rice leafhopper); Nilaparvatalugens Stil (brown planthopper); Peregrinus maidis Ashmead (cornplanthopper); Sogatellafurcifera Horvath (white-backed planthopper);Sogatodes orizicola Muir (rice delphacid); Typhlocyba pomaria McAtee(white apple leafhopper); Erythroneoura spp. (grape leafhoppers);Magicicada septendecim Linnaeus (periodical cicada); Icerya purchasiMaskell (cottony cushion scale); Quadraspidiotus perniciosus Comstock(San Jose scale); Planococcus citri Risso (citrus mealybug);Pseudococcus spp. (other mealybug complex); Cacopsylla pyricola Foerster(pear psylla); Trioza diospyri Ashmead (persimmon psylla).

Agronomically important species of interest from the order Hemipterainclude, but are not limited to: Acrosternum hilare Say (green stinkbug); Anasa tristis De Geer (squash bug); Blissus leucopterusleucopterus Say (chinch bug); Corythuca gossypii Fabricius (cotton lacebug); Cyrtopeltis modesta Distant (tomato bug); Dysdercus suturellusHerrich-Schaffer (cotton stainer); Euschistus servus Say (brown stinkbug); E. variolarius Palisot de Beauvois (one-spotted stink bug);Graptostethus spp. (complex of seed bugs); Leptoglossus corculus Say(leaf-footed pine seed bug); Lygus lineolaris Palisot de Beauvois(tarnished plant bug); L. Hesperus Knight (Western tarnished plant bug);L. pratensis Linnaeus (common meadow bug); L. rugulipennis Poppius(European tarnished plant bug); Lygocoris pabulinus Linnaeus (commongreen capsid); Nezara viridula Linnaeus (southern green stink bug);Oebalus pugnax Fabricius (rice stink bug); Oncopeltusfasciatus Dallas(large milkweed bug); Pseudatomoscelis seriatus Reuter (cottonfleahopper).

Furthermore, embodiments may be effective against Hemiptera such,Calocoris norvegicus Gmelin (strawberry bug); Orthops campestrisLinnaeus; Plesiocoris rugicollis Fallen (apple capsid); Cyrtopeltismodestus Distant (tomato bug); Cyrtopeltis notatus Distant (suckfly);Spanagonicus albofasciatus Reuter (whitemarked fleahopper); Diaphnocorischlorionis Say (honeylocust plant bug); Labopidicola allii Knight (onionplant bug); Pseudatomoscelis seriatus Reuter (cotton fleahopper);Adelphocoris rapidus Say (rapid plant bug); Poecilocapsus lineatusFabricius (four-lined plant bug); Nysius ericae Schilling (false chinchbug); Nysius raphanus Howard (false chinch bug); Nezara viridulaLinnaeus (Southern green stink bug); Eurygaster spp.; Coreidae spp.;Pyrrhocoridae spp.; Tinidae spp.; Blostomatidae spp.; Reduviidae spp.and Cimicidae spp.

Also included are adults and larvae of the order Acari (mites) such asAceria tosichella Keifer (wheat curl mite); Petrobia latens Müller(brown wheat mite); spider mites and red mites in the familyTetranychidae, Panonychus ulmi Koch (European red mite); Tetranychusurticae Koch (two spotted spider mite); (T. mcdanieli McGregor (McDanielmite); T. cinnabarinus Boisduval (carmine spider mite); T. turkestaniUgarov & Nikolski (strawberry spider mite); flat mites in the familyTenuipalpidae, Brevipalpus lewisi McGregor (citrus flat mite); rust andbud mites in the family Eriophyidae and other foliar feeding mites andmites important in human and animal health, i.e., dust mites in thefamily Epidermoptidae, follicle mites in the family Demodicidae, grainmites in the family Glycyphagidae, ticks in the order Ixodidae. Ixodesscapularis Say (deer tick); I. holocyclus Neumann (Australian paralysistick); Dermacentor variabilis Say (American dog tick); Amblyommaamericanum Linnaeus (lone star tick) and scab and itch mites in thefamilies Psoroptidae, Pyemotidae and Sarcoptidae.

Insect pest of interest include the superfamily of stink bugs and otherrelated insects including but not limited to species belonging to thefamily Pentatomidae (Nezara viridula, Halyomorpha halys, Piezodorusguildini, Euschistus servus, Acrosternum hilare, Euschistus heros,Euschistus tristigmus, Acrosternum hilare, Dichelops furcatus, Dichelopsmelacanthus, and Bagrada hilaris (Bagrada Bug)), the family Plataspidae(Megacopta cribraria—Bean plataspid) and the family Cydnidae(Scaptocoris castanea—Root stink bug) and Lepidoptera species includingbut not limited to: diamond-back moth, e.g., Helicoverpa zea Boddie;soybean looper, e.g., Pseudoplusia includens Walker and velvet beancaterpillar e.g., Anticarsia gemmatalis Hübner.

Methods for measuring pesticidal activity include for example, Czaplaand Lang, (1990) J. Econ. Entomol. 83:2480-2485; Andrews, et al., (1988)Biochem. J. 252:199-206; Marrone, et al., (1985) J. of EconomicEntomology 78:290-293 and U.S. Pat. No. 5,743,477, all of which areherein incorporated by reference in their entirety. Generally, theprotein is mixed and used in feeding assays. See, for example Marrone,et al., (1985) J. of Economic Entomology 78:290-293. Such assays caninclude contacting plants with one or more pests and determining theplant's ability to survive and/or cause the death of the pests.

Nematodes include parasitic nematodes such as root-knot, cyst and lesionnematodes, including Heterodera spp., Meloidogyne spp. and Globoderaspp.; particularly members of the cyst nematodes, including, but notlimited to, Heterodera glycines (soybean cyst nematode); Heteroderaschachtii (beet cyst nematode); Heterodera avenae (cereal cyst nematode)and Globodera rostochiensis and Globodera pailida (potato cystnematodes). Lesion nematodes include Pratylenchus spp.

Seed treatment. To protect and to enhance yield production and traittechnologies, seed treatment options can provide additional crop planflexibility and cost effective control against insects, weeds anddiseases. Seed material can be treated, typically surface treated, witha composition comprising combinations of chemical or biologicalherbicides, herbicide safeners, insecticides, fungicides, germinationinhibitors and enhancers, nutrients, plant growth regulators andactivators, bactericides, nematocides, avicides and/or molluscicides.These compounds are typically formulated together with further carriers,surfactants or application-promoting adjuvants employed in theformulation. The coatings may be applied by impregnating propagationmaterial with a liquid formulation or by coating with a combined wet ordry formulation. Examples of the various types of compounds that may beused as seed treatments are provided in The Pesticide Manual: A WorldCompendium, C. D. S. Tomlin Ed., Published by the British CropProduction Council, which is hereby incorporated by reference.

Some seed treatments that may be used on crop seed include, but are notlimited to, one or more of abscisic acid, acibenzolar-S-methyl,avermectin, amitrol, azaconazole, azospirillum, azadirachtin,azoxystrobin, Bacillus spp. (including one or more of cereus, firmus,megaterium, pumilis, sphaericus, subtilis and/or thuringiensis species),bradyrhizobium spp. (including one or more of betae, canariense,elkanii, iriomotense, japonicum, liaonigense, pachyrhizi and/oryuanmingense), captan, carboxin, chitosan, clothianidin, copper,cyazypyr, difenoconazole, etidiazole, fipronil, fludioxonil,fluoxastrobin, fluquinconazole, flurazole, fluxofenim, harpin protein,imazalil, imidacloprid, ipconazole, isoflavenoids,lipo-chitooligosaccharide, mancozeb, manganese, maneb, mefenoxam,metalaxyl, metconazole, myclobutanil, PCNB, penflufen, penicillium,penthiopyrad, permethrine, picoxystrobin, prothioconazole,pyraclostrobin, rynaxypyr, S-metolachlor, saponin, sedaxane, TCMTB,tebuconazole, thiabendazole, thiamethoxam, thiocarb, thiram,tolclofos-methyl, triadimenol, trichoderma, trifloxystrobin,triticonazole and/or zinc. PCNB seed coat refers to EPA RegistrationNumber 00293500419, containing quintozen and terrazole. TCMTB refers to2-(thiocyanomethylthio) benzothiazole.

Seed varieties and seeds with specific transgenic traits may be testedto determine which seed treatment options and application rates maycomplement such varieties and transgenic traits to enhance yield. Forexample, a variety with good yield potential but head smutsusceptibility may benefit from the use of a seed treatment thatprovides protection against head smut, a variety with good yieldpotential but cyst nematode susceptibility may benefit from the use of aseed treatment that provides protection against cyst nematode, and soon. Likewise, a variety encompassing a transgenic trait conferringinsect resistance may benefit from the second mode of action conferredby the seed treatment, a variety encompassing a transgenic traitconferring herbicide resistance may benefit from a seed treatment with asafener that enhances the plants resistance to that herbicide, etc.Further, the good root establishment and early emergence that resultsfrom the proper use of a seed treatment may result in more efficientnitrogen use, a better ability to withstand drought and an overallincrease in yield potential of a variety or varieties containing acertain trait when combined with a seed treatment.

Methods for killing an insect pest and controlling an insect population.In some embodiments, methods are provided for killing an insect pest,comprising contacting the insect pest, either simultaneously orsequentially, with an insecticidally-effective amount of a recombinantIRDIG35563 polypeptide of the disclosure. In some embodiments, methodsare provided for killing an insect pest, comprising contacting theinsect pest with an insecticidally-effective amount of a recombinantpesticidal protein of SEQ ID NO: 2 or a variant thereof.

In some embodiments, methods are provided for controlling an insect pestpopulation, comprising contacting the insect pest population, eithersimultaneously or sequentially, with an insecticidally-effective amountof a recombinant IRDIG35563 polypeptide of the disclosure. In someembodiments, methods are provided for controlling an insect pestpopulation, comprising contacting the insect pest population with aninsecticidally-effective amount of a recombinant IRDIG35563 polypeptideof SEQ ID NO: 2 or a variant thereof. As used herein, “controlling apest population” or “controls a pest” refers to any effect on a pestthat results in limiting the damage that the pest causes. Controlling apest includes, but is not limited to, killing the pest, inhibitingdevelopment of the pest, altering fertility or growth of the pest insuch a manner that the pest provides less damage to the plant,decreasing the number of offspring produced, producing less fit pests,producing pests more susceptible to predator attack or deterring thepests from eating the plant.

In some embodiments, methods are provided for controlling an insect pestpopulation resistant to a pesticidal protein, comprising contacting theinsect pest population, either simultaneously or sequentially, with aninsecticidally-effective amount of a recombinant IRDIG35563 polypeptideof the disclosure. In some embodiments, methods are provided forcontrolling an insect pest population resistant to a pesticidal protein,comprising contacting the insect pest population with aninsecticidally-effective amount of a recombinant IRDIG35563 polypeptideof SEQ ID NO: 2 or a variant thereof.

In some embodiments, methods are provided for protecting a plant from aninsect pest, comprising expressing in the plant or cell thereof at leastone recombinant polynucleotide encoding an IRDIG35563 polypeptide. Insome embodiments, methods are provided for protecting a plant from aninsect pest, comprising expressing in the plant or cell thereof arecombinant polynucleotide encoding IRDIG35563 polypeptide of SEQ ID NO:2 or variants thereof.

Insect resistance management (IRM) strategies. Expression of B.thuringiensis δ-endotoxins in transgenic corn plants has proven to be aneffective means of controlling agriculturally important insect pests(Perlak, et al., 1990; 1993). However, insects have evolved that areresistant to B. thuringiensis δ-endotoxins expressed in transgenicplants. Such resistance, should it become widespread, would clearlylimit the commercial value of germplasm containing genes encoding suchB. thuringiensis δ-endotoxins.

One way to increasing the effectiveness of the transgenic insecticidesagainst target pests and contemporaneously reducing the development ofinsecticide-resistant pests is to use provide non-transgenic (i.e.,non-insecticidal protein) refuges (a section of non-insecticidalcrops/corn) for use with transgenic crops producing a singleinsecticidal protein active against target pests. The United StatesEnvironmental Protection Agency(epa.gov/oppbppdl/biopesticides/pips/bt_corn_refuge_2006.htm, which canbe accessed using the www prefix) publishes the requirements for usewith transgenic crops producing a single Bt protein active againsttarget pests. In addition, the National Corn Growers Association, ontheir website:(ncga.com/insect-resistance-management-fact-sheet-bt-corn, which can beaccessed using the www prefix) also provides similar guidance regardingrefuge requirements. Due to losses to insects within the refuge area,larger refuges may reduce overall yield.

Another way of increasing the effectiveness of the transgenicinsecticides against target pests and contemporaneously reducing thedevelopment of insecticide-resistant pests would be to have a repositoryof insecticidal genes that are effective against groups of insect pestsand which manifest their effects through different modes of action.

Expression in a plant of two or more insecticidal compositions toxic tothe same insect species, each insecticide being expressed at efficaciouslevels would be another way to achieve control of the development ofresistance. This is based on the principle that evolution of resistanceagainst two separate modes of action is far more unlikely than only one.Roush, for example, outlines two-toxin strategies, also called“pyramiding” or “stacking,” for management of insecticidal transgeniccrops. (The Royal Society. Phil. Trans. R. Soc. Lond. B. (1998)353:1777-1786). Stacking or pyramiding of two different proteins eacheffective against the target pests and with little or nocross-resistance can allow for use of a smaller refuge. The USEnvironmental Protection Agency requires significantly less (generally5%) structured refuge of non-Bt corn be planted than for single traitproducts (generally 20%). There are various ways of providing the IRMeffects of a refuge, including various geometric planting patterns inthe fields and in-bag seed mixtures, as discussed further by Roush.

In some embodiments, the IRDIG35563 polypeptides of the disclosure areuseful as an insect resistance management strategy in combination (i.e.,pyramided) with other pesticidal proteins include but are not limited toBt toxins, Xenorhabdus sp. or Photorhabdus sp. insecticidal proteins,other insecticidally active proteins, and the like.

Provided are methods of controlling Lepidoptera and/or Coleoptera insectinfestation(s) in a transgenic plant that promote insect resistancemanagement, comprising expressing in the plant at least two differentinsecticidal proteins having different modes of action.

In some embodiments, the methods of controlling Lepidoptera and/orColeoptera insect infestation in a transgenic plant and promoting insectresistance management comprises the presentation of at least one of theIRDIG35563 polypeptide insecticidal proteins to insects in the orderLepidoptera and/or Coleoptera.

In some embodiments, the methods of controlling Lepidoptera and/orColeoptera insect infestation in a transgenic plant and promoting insectresistance management comprises the presentation of at least one of theIRDIG35563 polypeptides of SEQ ID NO: 2, or variants thereof,insecticidal to insects in the order Lepidoptera and/or Coleoptera.

In some embodiments, the methods of controlling Lepidoptera and/orColeoptera insect infestation in a transgenic plant and promoting insectresistance management comprise expressing in the transgenic plant anIRDIG35563 polypeptide and a Cry protein or other insecticidal proteinto insects in the order Lepidoptera and/or Coleoptera having differentmodes of action.

In some embodiments, the methods, of controlling Lepidoptera and/orColeoptera insect infestation in a transgenic plant and promoting insectresistance management, comprise expression in the transgenic plant anIRDIG35563 polypeptide of SEQ ID NO: 2 or variants thereof and a Cryprotein or other insecticidal protein to insects in the orderLepidoptera and/or Coleoptera, where the IRDIG35563 polypeptide and Cryprotein have different modes of action.

Also provided are methods of reducing likelihood of emergence ofLepidoptera and/or Coleoptera insect resistance to transgenic plantsexpressing in the plants insecticidal proteins to control the insectspecies, comprising expression of an IRDIG35563 polypeptide insecticidalto the insect species in combination with a second insecticidal proteinto the insect species having different modes of action.

Also provided are means for effective Lepidoptera and/or Coleopterainsect resistance management of transgenic plants, comprisingco-expressing at high levels in the plants two or more insecticidalproteins toxic to Lepidoptera and/or Coleoptera insects but eachexhibiting a different mode of effectuating its killing activity,wherein the two or more insecticidal proteins comprise an IRDIG35563polypeptide and a Cry protein. Also provided are means for effectiveLepidoptera and/or Coleoptera insect resistance management of transgenicplants, comprising co-expressing at high levels in the plants two ormore insecticidal proteins toxic to Lepidoptera and/or Coleopterainsects but each exhibiting a different mode of effectuating its killingactivity, wherein the two or more insecticidal proteins comprise anIRDIG35563 polypeptide of SEQ ID NO: 2 or variants thereof and a Cryprotein or other insecticidally active protein.

In addition, methods are provided for obtaining regulatory approval forplanting or commercialization of plants expressing proteins insecticidalto insects in the order Lepidoptera and/or Coleoptera, comprising thestep of referring to, submitting or relying on insect assay binding datashowing that the IRDIG35563 polypeptide does not compete with bindingsites for Cry proteins in such insects. In addition, methods areprovided for obtaining regulatory approval for planting orcommercialization of plants expressing proteins insecticidal to insectsin the order Lepidoptera and/or Coleoptera, comprising the step ofreferring to, submitting or relying on insect assay binding data showingthat the IRDIG35563 polypeptide of SEQ ID NO: 2, or variant thereof doesnot compete with binding sites for Cry proteins in such insects.

Methods for increasing plant yield. The methods comprise providing aplant or plant cell expressing a polynucleotide encoding the pesticidalpolypeptide sequence disclosed herein and growing the plant or a seedthereof in a field infested with a pest against which the polypeptidehas pesticidal activity. In some embodiments, the polypeptide haspesticidal activity against a Lepidopteran, Coleopteran, Dipteran,Hemipteran or nematode pest, and the field is infested with aLepidopteran, Hemipteran, Coleopteran, Dipteran or nematode pest.

As defined herein, the “yield” of the plant refers to the quality and/orquantity of biomass produced by the plant. “Biomass” as used hereinrefers to any measured plant product. An increase in biomass productionis any improvement in the yield of the measured plant product.Increasing plant yield has several commercial applications. For example,increasing plant leaf biomass may increase the yield of leafy vegetablesfor human or animal consumption. Additionally, increasing leaf biomasscan be used to increase production of plant-derived pharmaceutical orindustrial products. An increase in yield can comprise any statisticallysignificant increase including, but not limited to, at least a 1%increase, at least a 3% increase, at least a 5% increase, at least a 10%increase, at least a 20% increase, at least a 30%, at least a 50%, atleast a 70%, at least a 100% or a greater increase in yield compared toa plant not expressing the pesticidal sequence.

In specific methods, plant yield is increased as a result of improvedpest resistance of a plant expressing an IRDIG35563 polypeptidedisclosed herein. Expression of the IRDIG35563 polypeptide results in areduced ability of a pest to infest or feed on the plant, thus improvingplant yield.

Methods of processing. Further provided are methods of processing aplant, plant part or seed to obtain a food or feed product from a plant,plant part or seed comprising an IRDIG35563 polynucleotide. The plants,plant parts or seeds provided herein, can be processed to yield oil,protein products and/or by-products that are derivatives obtained byprocessing that have commercial value. Non-limiting examples includetransgenic seeds comprising a nucleic acid molecule encoding anIRDIG35563 polypeptide which can be processed to yield soy oil, soyproducts and/or soy by-products.

“Processing” refers to any physical and chemical methods used to obtainany soy product and includes, but is not limited to, heat conditioning,flaking and grinding, extrusion, solvent extraction or aqueous soakingand extraction of whole or partial seeds.

When an insect comes into contact and ingests an effective amount oftoxin delivered via transgenic plant expression, formulated proteincomposition(s), sprayable protein composition(s), a bait matrix or otherdelivery system, the results are typically death of the insect, or theinsects do not feed upon the source which makes the toxins available tothe insects.

With suitable microbial hosts, e.g. Pseudomonas, the microbes can beapplied to the environment of the pest, where they will proliferate andbe ingested. The result is control of the pest. Alternatively, themicrobe hosting the toxin gene can be treated under conditions thatprolong the activity of the toxin and stabilize the cell. The treatedcell, which retains the toxic activity, then can be applied to theenvironment of the target pest.

EXAMPLES Example 1

Construction of expression plasmids encoding IRDIG35563 insecticidaltoxin and expression in bacterial hosts. Standard cloning methods wereused in the construction of Pseudomonas fluorescens (Pf) expressionplasmids engineered to produce IRDIG35563 proteins encoded byplant-optimized coding regions. Restriction endonucleases and T4 DNALigase were obtained from New England BioLabs (NEB; Ipswich, Mass.) forrestriction digestion and DNA ligation, respectively. Plasmidpreparations were performed using the NucleoSpin® Plasmid Kit(Macherey-Nagel Inc, Bethlehem, Pa.) following the instructions of thesuppliers for low-copy plasmid purification. DNA fragments were purifiedusing a QIAquick® Gel Extraction Kit (Qiagen, Venio, Limburg) afteragarose Tris-acetate gel electrophoresis.

The basic cloning strategy entailed subcloning the IRDIG35563 toxincoding sequence (CDS) (SEQ ID NO:1) into pDOW1169 at the Spel and Salrestriction sites, whereby it is placed under the expression control ofthe Ptac promoter and the rrnBT1T2 terminator from plasmid pKK223-3 (PLPharmacia, Milwaukee, Wis.). pDOW1169 is a medium copy plasmid with theRSF1010 origin of replication, apyrF gene, and a ribosome binding sitepreceding the restriction enzyme recognition sites into which DNAfragments containing protein coding regions may be introduced, (USApplication 20080193974). The expression plasmid, designated pDOW1169,was transformed by electroporation into DC454 (a near wild-type P.fluorescens strain having mutations deltapyrF and lsc::lacI^(QI)), orits derivatives, recovered in SOC-Soy hydrolysate medium, and plated onselective medium (M9 glucose agar lacking uracil, Sambrook et al.,supra). Details of the microbiological manipulations are available inSquires et al., (2004), US Patent Application 20060008877, US PatentApplication 20080193974, and US Patent Application 20080058262,incorporated herein by reference. Colonies were first screened byrestriction digestion of miniprep plasmid DNA. Plasmid DNA of selectedclones containing IRDIG35563 toxin were digested with four restrictionenzymes and sequence verified to further validate presence of theinsert.

Example 2

Growth and Expression Analysis in Shake Flasks. Production of IRDIG35563toxin for characterization and insect bioassay was accomplished byshake-flask-grown P. fluorescens strains harboring expression constructs(pDOW1169). A glycerol stock of IRDIG35563 culture (0.5 mL) wasinoculated into 50 mL of defined production medium with 9.5% glycerol(Teknova Catalog No. 3D7426, Hollister, Calif.). Expression of theIRDIG35563 toxin gene via the Ptac promoter was induced by addition ofisopropyl-β-D-1-thiogalactopyranoside (IPTG) after an initial incubationof 24 hours at 30° C. with shaking. Cultures were sampled at the time ofinduction and at various times post-induction. Cell density was measuredby optical density at 600 nm (OD₆₀₀). Other culture media suitable forgrowth of Pseudomonas fluorescens may also be utilized, for example, asdescribed US Patent Application 20060008877.

Example 3

Agrobacterium transformation. Standard cloning methods were used in theconstruction of binary plant transformation and expression plasmids.Restriction endonucleases and T4 DNA Ligase were obtained from NewEngland Biolabs. Plasmid preparations were performed using theNucleoSpin® Plasmid Preparation kit or the NucleoBond® AX Xtra Midi kit(both from Macherey-Nagel, Duren, Germany), following the instructionsof the manufacturers. DNA fragments were purified using the QIAquick®PCR Purification Kit or the QIAEX II® Gel Extraction Kit (both fromQiagen Venio, Limburg) after gel isolation.

Electro-competent cells of Agrobacterium tumefaciens strain Z707S (astreptomycin-resistant derivative of Z707; Hepburn et al., 1985) wereprepared and transformed using electroporation (Weigel and Glazebrook,2002). After electroporation, 1 mL of Yeast Extract Peptone (YEP) broth(10 gm/L yeast extract, 10 gm/L peptone, and 5 gm/L NaCl) was added tothe cuvette and the cell-YEP suspension was transferred to a 15 mLculture tube for incubation at 28° C. in a water bath with constantagitation for 4 hours. The cells were plated on YEP plus agar (25 gm/L)with spectinomycin (200 μg/mL) and streptomycin (250 μg/mL) and theplates were incubated for 2-4 days at 28° C. Well separated singlecolonies were selected and streaked onto fresh YEP+agar plates withspectinomycin and streptomycin as described above, and incubated at 28°C. for 1-3 days.

The presence of the IRDIG35563 gene insert in the binary planttransformation vector was performed by PCR analysis usingvector-specific primers with template plasmid DNA prepared from selectedAgrobacterium colonies. The cell pellet from a 4 mL aliquot of a 15 mLovernight culture grown in YEP with spectinomycin and streptomycin asbefore was extracted using Qiagen (Venlo, Limburg, Netherlands) Spin®Mini Preps, performed per manufacturer's instructions. Plasmid DNA fromthe binary vector used in the Agrobacterium electroporationtransformation was included as a control. The PCR reaction was completedusing Taq DNA polymerase from Invitrogen (Carlsbad, Calif.) permanufacturer's instructions at 0.5× concentrations. PCR reactions werecarried out in a MJ Research Peltier Thermal Cycler programmed with thefollowing conditions: Step 1) 94° C. for 3 minutes; Step 2) 94° C. for45 seconds; Step 3) 55° C. for 30 seconds; Step 4) 72° C. for 1 minuteper kb of expected product length; Step 5) 29 times to Step 2; Step 6)72° C. for 10 minutes. The reaction was maintained at 4° C. aftercycling. The amplification products were analyzed by agarose gelelectrophoresis (e.g. 0.7% to 1% agarose, w/v) and visualized byethidium bromide staining. A colony was selected whose PCR product wasidentical to the plasmid control.

Example 4

IRDIG35563 purification. Harvested Pf cells containing IRDIG35563 weresonicated in lysis buffer consisting of 50 mM Tris (pH 8.0), 1 M NaCl,10% glycerol and 2 mM EDTA with 50 μL of Protease Inhibitor Cocktail(Sigma-Aldrich, St. Louis, Mo.) per 25 mL buffer. The extract wascentrifuged at 20,000×g for 40 minutes. The soluble protein in thesupernatant was precipitated with 50% ammonium sulfate and centrifugedat 20,000×g for 30 minutes. The pellet was resuspended in 50 mM Tris (pH8.0) and centrifuged at 20,000×g for 20 minutes to pellet any solublematerial. The supernatant containing IRDIG35563 was purified by anionexchange chromatography using a HiTrap™ Q HP 5 mL column with an AKTAPurifier chromatography system (GE Healthcare, UK). The column wasequilibrated in 50 mM Tris (pH 8.0), and proteins were eluted with astepwise gradient to 1 M NaCl. Protein-containing fractions werecombined and concentrated using Amicon® Ultra-15 Centrifugal FilterDevices with a 30K MWCO (EMD Millipore, Burlington, Mass.). TheIRDIG35563 protein sample was dialyzed overnight against 50 mM Tris (pH8.), and total protein concentrations were measured with the NanoDrop2000C Spectrophotometer (Thermo Scientific, Waltham, Mass.), using theA280 method.

Example 5

Gel electrophoresis. SDS-PAGE analysis was performed using NuPAGE®Novex® 4-12% Bis-Tris Protein Gels (Thermo Scientific, Waltham, Mass.).Proteins were diluted 4× in NuPAGE® LDS Sample Buffer (ThermoScientific, Waltham, Mass.) containing 100 mM TCEP prior to loading ontothe gel. Ten μL of Novex® Sharp Pre-stained Protein Standard was loadedonto one lane of each gel. Gels were run in NuPAGE® MES SDS RunningBuffer according to the manufacturer's recommendations and stained withSimplyBlue™ SafeStain (Thermo Scientific, Waltham, Mass.), thendestained in water and imaged on a flatbed scanner. Final proteinpreparation showed a single band of protein migrating at an apparentmolecular weight of about 88 kDa.

Example 6

Sample preparation and bioassays. Purified preparations of IRDIG35563were diluted appropriately in 50 mM Tris, pH 8, and all bioassayscontained a control treatment consisting of this buffer, which served asa background check for mortality and/or growth inhibition. Proteinconcentrations in bioassay buffer were estimated with the NanoDrop 2000CSpectrophotometer (Thermo Scientific, Waltham, Mass.), using the A280method.

Purified proteins were tested for insecticidal activity in bioassaysconducted with 1-2 day old neonate Lepidopteran larvae on artificialinsect diet. Larvae of BAW, SAW, FAW, rFAW, CEW, TBW, SBL, VBC, and CBWwere hatched from eggs obtained from a colony maintained by a commercialinsectary (Benzon Research Inc., Carlisle, Pa.). Larvae of rFAW werehatched from eggs harvested from proprietary colonies (Dow AgroSciencesLLC, Indianapolis, Ind.).

The bioassays were conducted in 128-well plastic trays specificallydesigned for insect bioassays (C-D International, Pitman, N.J.). Eachwell contained 2.0 mL of Multi-species Lepidoptera diet (SouthlandProducts, Lake Village, Ark.). A 40 μL aliquot of protein sample wasdelivered by pipette onto the 2.0 cm² diet surface of each well (20μL/cm²). Diet concentrations were calculated as the amount (ng) ofIRDIG35563 protein per square centimeter (cm²) of surface area in thewell. A 9 dose concentration range was used from 9,000 to 3 ng/cm² with16 larvae tested per dose. Helicoverpa armigera bioassays used 7different concentrations of purified protoxins, with a control of onlybuffer. Mortality and arrest were assessed after 7 days at 25° C. with16:8 light:dark conditions. The treated trays were held in a fume hooduntil the liquid on the diet surface had evaporated or was absorbed intothe diet.

Within 24-48 hours of eclosion, individual larvae were picked up with amoistened camel hair brush and deposited on the treated diet, one larvaper well. The infested wells were then sealed with adhesive sheets ofclear plastic and vented to allow gas exchange (C-D International,Pitman, N.J.). Bioassay trays were held under controlled environmentalconditions (28° C., ˜60% Relative Humidity, 16:8 [Light:Dark]) for 5days, after which the total number of insects exposed to each proteinsample, the number of dead insects, and the weight of surviving insectswere recorded.

The GI₅₀ was determined to be the concentration of IRDIG35563 protein inthe diet at which the GI value was 50%. The 50% lethal concentration(LC₅₀) was recorded as the concentration of IRDIG35563 protein in thediet at which 50% of test insects were killed. Growth inhibitionconcentration-response curves were determined by using a nonlinearlogistic 3-parameter through JMP Pro, version 9.0.3, software (SASInstitute Inc., Cary, N.C.). Lethal concentration-response curve wereanalyzed by Probit analyses (Finney, 1971) of the pooled mortality dataand were conducted using POLO-PC (LeOra Software). Table 2 shows theefficacy of purified IRDIG35563 against multiple insect species testedon artificial diet-overlay bioassay (ng/cm²).

TABLE 2 % mortality Practical mortality Target Insect N (3000 ng/cm²)(3000 ng/cm²) BAW 16 100.0 100.0 CBW 16 100.0 100.0 CEW 46 21.7 65.2 ECB16 6.3 12.5 FAW 32 93.8 100 rFAW (Cry1F resistant) 32 87.5 100 SAW 32100.0 100.0 SBL 32 40.6 50.0 TBW 16 37.5 50.0 VBC 16 62.5 100.0

Example 7 Production of IRDIG35563 Bt Insecticidal Proteins and Variantsin Dicot Plants

Arabidopsis Transformation. Arabidopsis thaliana Col-01 was transformedusing the floral dip method (Weigel and Glazebrook, 2002). The selectedAgrobacterium colony was used to inoculate 1 mL to 15 mL cultures of YEPbroth containing appropriate antibiotics for selection. The culture wasincubated overnight at 28° C. with constant agitation at 220 rpm. Eachculture was used to inoculate two 500 mL cultures of YEP brothcontaining appropriate antibiotics for selection and the new cultureswere incubated overnight at 28° C. with constant agitation. The cellswere pelleted at approximately 8700×g for 10 minutes at roomtemperature, and the resulting supernatant was discarded. The cellpellet was gently resuspended in 500 mL of infiltration mediacontaining: ½×Murashige and Skoog salts (Sigma-Aldrich)/Gamborg's B5vitamins (Gold BioTechnology, St. Louis, Mo.), 10% (w/v) sucrose, 0.044μM benzylaminopurine (10 μL/L of 1 mg/mL stock in DMSO) and 300 μL/LSilwet L-77. Plants approximately 1 month old were dipped into the mediafor 15 seconds, with care taken to assure submergence of the newestinflorescence. The plants were then laid on their sides and covered(transparent or opaque) for 24 hours, washed with water, and placedupright. The plants were grown at 22° C., with a 16:8 light:darkphotoperiod. Approximately 4 weeks after dipping, the seeds wereharvested.

Arabidopsis Growth and Selection. Freshly harvested T1 seed was allowedto dry for at least 7 days at room temperature in the presence ofdesiccant. Seed was suspended in a 0.1% agar/water (Sigma-Aldrich)solution and then stratified at 4° C. for 2 days. To prepare forplanting, Sunshine Mix LP5 (Sun Gro Horticulture Inc., Bellevue, Wash.)in 10.5 inch×21 inch germination trays (T.O. Plastics Inc., Clearwater,Minn.) was covered with fine vermiculite, sub-irrigated with Hoagland'ssolution (Hoagland and Arnon, 1950) until wet, then allowed to drain for24 hours. Stratified seed was sown onto the vermiculite and covered withhumidity domes (KORD Products, Bramalea, Ontario, Canada) for 7 days.Seeds were germinated and plants were grown in a Conviron (ModelsCMP4030 or CMP3244; Controlled Environments Limited, Winnipeg, Manitoba,Canada) under long day conditions (16:8 light:dark photoperiod) at alight intensity of 120-150 μmol/m² sec under constant temperature (22°C.) and humidity (40-50%). Plants were initially watered with Hoagland'ssolution and subsequently with deionized water to keep the soil moistbut not wet.

The domes were removed 5-6 days post sowing and plants were sprayed witha chemical selection agent to kill plants germinated from nontransformedseeds. For example, if the plant expressible selectable marker geneprovided by the binary plant transformation vector is apat or bar gene(Wehrmann et al., 1996), transformed plants may be selected by sprayingwith a 1000× solution of Finale (5.78% glufosinate ammonium, FarnamCompanies Inc., Phoenix, Ariz.). Two subsequent sprays were performed at5-7 day intervals. Survivors (plants actively growing) were identified7-10 days after the final spraying and were transplanted into potsprepared with Sunshine Mix LP5. Transplanted plants were covered with ahumidity dome for 3-4 days and placed in a Conviron under theabove-mentioned growth conditions.

Those skilled in the art of dicot plant transformation will understandthat other methods of selection of transformed plants are available whenother plant expressible selectable marker genes (e.g. herbicidetolerance genes) are used. Table 3 shows the results (average score(SEM)) of Arabidopsis T1 bioassay against CEW, FAW, SAW, TBW, and SBL.Five events were generated for each construct and the average leafdamage score (5 leaf punches/event, 5 days post infestation) wasrecorded 5 weeks after germination. Construct design and numbering arereported in Table 4.

TABLE 3 Protein Construct CEW FAW SAW TBW SBL IRDIG35563 pDAB134633 0.46(0.22) 0.42 (0.19) 0.35 (0.15) 0.45 (0.21) 0.40 (0.19) pDAB134634 0.1(0)   0.1 (0)   0.1 (0)   0.11 (0.01) 0.1 (0)   pDAB134635 0.1 (0)   0.1(0)   0.1 (0)   0.30 (0.03) 0.1 (0)   pDAB134636 0.1 (0)   0.1 (0)   0.1(0)   0.11 (0.01) 0.1 (0)   Vip3Ab1 Pos Control 0.11 (0.01) 0.1 (0)  0.74 (0.06) 0.1 (0)   0.1 (0)   Col-0 Neg Control 1 (0) 0.82 (0.07) 0.61(0.08) 0.93 (0.04) 0.98 (0.03)

TABLE 4 pDAB134633 Agro Vector with AtUbi10 :: IRDIG35563.2 :: AtUbi10 +ZC21 + CsVMV :: DSM2 :: Orf1 pDAB134634 Agro Vector with AtUbi3 ::IRDIG35563.2 :: AtUbi3 + ZC21 + CsVMV :: DSM2 :: Orf1 pDAB134635 AgroVector with AtAct2 :: IRDIG35563.2 :: AtAct2 + ZC21 + CsVMV :: DSM2 ::Orf1 pDAB134636 Agro Vector with GmCAB :: IRDIG35563.2 :: GmCAB + ZC21 +CsVMV :: DSM2 :: Orf1

Example 8 Transgenic Glycine max Comprising IRDIG35563

Ten to 20 transgenic T₀ Glycine max plants harboring expression vectorsfor nucleic acids comprising IRDIG35563 are generated, for example, byAgrobacterium-mediated transformation. Mature soybean (Glycine max)seeds are sterilized overnight with chlorine gas for sixteen hours.Following sterilization with chlorine gas, the seeds are placed in anopen container in a LAMINAR™ flow hood to dispel the chlorine gas. Next,the sterilized seeds are imbibed with sterile H₂O for sixteen hours inthe dark using a black box at 24° C.

Preparation of split-seed soybeans. The split soybean seed comprising aportion of an embryonic axis protocol requires preparation of soybeanseed material which is cut longitudinally, using a #10 blade affixed toa scalpel, along the hilum of the seed to separate and remove the seedcoat, and to split the seed into two cotyledon sections. Carefulattention is made to partially remove the embryonic axis, wherein about½-⅓ of the embryo axis remains attached to the nodal end of thecotyledon.

Inoculation. The split soybean seeds comprising a partial portion of theembryonic axis are then immersed for about 30 minutes in a solution ofAgrobacterium tumefaciens (e.g., strain EHA 101 or EHA 105) containingbinary plasmid comprising IRDIG35563. The Agrobacterium tumefacienssolution is diluted to a final concentration of λ=0.6 OD₆₅₀ beforeimmersing the cotyledons comprising the embryo axis.

Co-cultivation. Following inoculation, the split soybean seed is allowedto co-cultivate with the Agrobacterium tumefaciens strain for 5 days onco-cultivation medium (Wang, Kan. Agrobacterium Protocols. 2. 1. NewJersey: Humana Press, 2006. Print.) in a Petri dish covered with a pieceof filter paper.

Shoot induction. After 5 days of co-cultivation, the split soybean seedsare washed in liquid Shoot Induction (SI) media consisting of B5 salts,B5 vitamins, 28 mg/L Ferrous, 38 mg/L Na₂EDTA, 30 g/L sucrose, 0.6 g/LMES, 1.11 mg/L BAP, 100 mg/L TIMENTIN™ 200 mg/L cefotaxime, and 50 mg/Lvancomycin (pH 5.7). The split soybean seeds are then cultured on ShootInduction I (SI I) medium consisting of B5 salts, B5 vitamins, 7 g/LNoble agar, 28 mg/L Ferrous, 38 mg/L Na₂EDTA, 30 g/L sucrose, 0.6 g/LMES, 1.11 mg/L BAP, 50 mg/L TIMENTIN™, 200 mg/L cefotaxime, 50 mg/Lvancomycin (pH 5.7), with the flat side of the cotyledon facing up andthe nodal end of the cotyledon imbedded into the medium. After 2 weeksof culture, the explants from the transformed split soybean seed aretransferred to the Shoot Induction II (SI II) medium containing SI Imedium supplemented with 6 mg/L glufosinate (LIBERTY®).

Shoot elongation. After 2 weeks of culture on SI II medium, thecotyledons are removed from the explants and a flush shoot padcontaining the embryonic axis are excised by making a cut at the base ofthe cotyledon. The isolated shoot pad from the cotyledon is transferredto Shoot Elongation (SE) medium. The SE medium consists of MS salts, 28mg/L Ferrous, 38 mg/L Na₂EDTA, 30 g/L sucrose and 0.6 g/L MES, 50 mg/Lasparagine, 100 mg/L L-pyroglutamic acid, 0.1 mg/L IAA, 0.5 mg/L GA3, 1mg/L zeatin riboside, 50 mg/L TIMENTIN™, 200 mg/L cefotaxime, 50 mg/Lvancomycin, 6 mg/L glufosinate, 7 g/L Noble agar, (pH 5.7). The culturesare transferred to fresh SE medium every 2 weeks. The cultures are grownin a CONVIRON™ growth chamber at 24° C. with an 18 h photoperiod at alight intensity of 80-90 μmol/m² sec.

Rooting. Elongated shoots which developed from the cotyledon shoot padare isolated by cutting the elongated shoot at the base of the cotyledonshoot pad, and dipping the elongated shoot in 1 mg/L IBA (Indole3-butyric acid) for 1-3 minutes to promote rooting. Next, the elongatedshoots are transferred to rooting medium (MS salts, B5 vitamins, 28 mg/LFerrous, 38 mg/L Na₂EDTA, 20 g/L sucrose and 0.59 g/L MES, 50 mg/Lasparagine, 100 mg/L L-pyroglutamic acid 7 g/L Noble agar, pH 5.6) inphyta trays.

Cultivation. Following culture in a CONVIRON™ growth chamber at 24° C.,18 h photoperiod, for 1-2 weeks, the shoots which have developed rootsare transferred to a soil mix in a covered sundae cup and placed in aCONVIRON™ growth chamber (models CMP4030 and CMP3244, ControlledEnvironments Limited, Winnipeg, Manitoba, Canada) under long dayconditions (16 hours light/8 hours dark) at a light intensity of 120-150μmol/m² sec under constant temperature (22° C.) and humidity (40-50%)for acclimatization of plantlets. The rooted plantlets are acclimated insundae cups for several weeks before they are transferred to thegreenhouse for further acclimatization and establishment of robusttransgenic soybean plants.

Development and morphological characteristics of transgenic lines arecompared with nontransformed plants. Plant root, shoot, foliage andreproduction characteristics are compared. Plant shoot characteristicssuch as height, leaf numbers and sizes, time of flowering, floral sizeand appearance are recorded. In general, there are no observablemorphological differences between transgenic lines and those withoutexpression of DIG proteins when cultured in vitro and in soil in theglasshouse.

Example 9

T0 Bioassays: Five soybean events were generated for each construct andthe average leaf damage score (5 leaf punches/event, 5 days postinfestation) was recorded 5 weeks after germination. Construct designand numbering are reported in Table 4. Table 5 shows the results ofsoybean T0 bioassay against CEW, FAW, SAW, TBW, and SBL. The leaf damagegrading scale utilizes and scoring system of 1 through 9, with 1representing 88-100% damage. Deregulated transgenic soybean, Conkestawas used as a positive control. A non-transgenic soybean line was usedas a negative control. The results for those events for which proteinwas detectable are presented in Table 5. Other test events producedinconclusive results due to, it is believed, environmental conditionsexperienced during transport. Table 6 presents the damage scalerepresented in Table 5.

TABLE 5 Event Feature (RE) BCW CBW CEW FAW SAW SBL TBW VBC Negativecontrol Negative 1 3 1 1 1 1 1 1 Negative control Negative 1 5 1 1 1 1 11 Negative control Negative 1 5 1 1 5 2 1 1 Negative control Negative 19 1 1 6.2 3 1 2 Positive control Positive 1 8 8 9 7 9 9 9 Positivecontrol Positive 1 9 9 9 8 9 9 9 Positive control Positive 3 9 9 9 8 9 99 Positive control Positive 3 9 9 9 9 9 9 9 134635[8]-254 AtActin2 7 9 79 9 9 4 9 134633[9]-209 AtUbi10 8 8 9 9 8 9 9 9 134633[10]-224 AtUbi10 85 7 9 9 9 4 8 134633[10]-212 AtUbi10 5 8 8 9 6.2 9 5 8

TABLE 6 Damage scale Percent damage 9 0 8 12.5 7 25 6.2 35 5 50 4 62.5 375 2 87.5 1 100

Example 10 Production of IRDIG35563

Agrobacterium-Mediated Transformation of Maize. Seeds from a High II orB-104 F₁ cross (Armstrong et al., 1991) were planted into 5-gallon-potscontaining a mixture of 95% Metro-Mix 360 soilless growing medium (SunGro Horticulture, Bellevue, Wash.) and 5% clay/loam soil. The plantswere grown in a greenhouse using a combination of high pressure sodiumand metal halide lamps with a 16:8 hour light:dark photoperiod. Toobtain immature F₂ embryos for transformation, controlledsib-pollinations were performed. Immature embryos were isolated at 8-10days post-pollination when embryos were approximately 1.0 to 2.0 mm insize.

Infection and co-cultivation Maize ears were surface sterilized byscrubbing with liquid soap, immersing in 70% ethanol for 2 minutes, andthen immersing in 20% commercial bleach (0.1% sodium hypochlorite) for30 minutes before being rinsed with sterile water. A suspension ofAgrobacterium cells containing a superbinary vector were prepared bytransferring 1-2 loops of bacteria grown on YEP solid medium containing100 mg/L spectinomycin, 10 mg/L tetracycline, and 250 mg/L streptomycinat 28° C. for 2-3 days into 5 mL of liquid infection medium (LS BasalMedium (Linsmaier and Skoog, 1965), N6 vitamins (Chu et al., 1975), 1.5mg/L 2,4-Dichlorophenoxyacetic acid (2,4-D), 68.5 gm/L sucrose, 36.0gm/L glucose, 6 mM L-proline, pH 5.2) containing 100 μM acetosyringone.The solution was vortexed until a uniform suspension was achieved, andthe concentration was adjusted to a final density of about 200 Klettunits, using a Klett-Summerson colorimeter with a purple filter, or anoptical density of approximately 0.4 at 550 nm. Immature embryos wereisolated directly into a micro centrifuge tube containing 2 mL of theinfection medium. The medium was removed and replaced with 1 mL of theAgrobacterium solution with a density of 200 Klett units, and theAgrobacterium and embryo solution was incubated for 5 minutes at roomtemperature and then transferred to co-cultivation medium (LS BasalMedium, (containing N6 vitamins, 1.5 mg/L 2,4-D, 30.0 gm/L sucrose, 6 mML-proline, 0.85 mg/L AgNO₃, 100 μM acetosyringone, and 3.0 gm/L Gellangum (PhytoTechnology Laboratories., Lenexa, Kans.), pH 5.8) for 5 daysat 25° C. under dark conditions.

After co-cultivation, the embryos were transferred to selective mediumafter which transformed isolates were obtained over the course ofapproximately 8 weeks. For selection of maize tissues transformed with asuperbinary plasmid containing a plant expressible pat or bar selectablemarker gene, an LS based medium (LS Basal medium, with 1×N6 vitamins,1.5 mg/L 2,4-D, 0.5 gm/L MES (2-(N-morpholino)ethanesulfonic acidmonohydrate; PhytoTechnology Laboratories., Lenexa, Kans.), 30.0 gm/Lsucrose, 6 mM L-proline, 1.0 mg/L AgNO₃, 250 mg/L cefotaxime, 2.5 gm/LGellan gum, pH 5.7) was used with Bialaphos (Gold BioTechnology, St.Louis, Mo.). The embryos were transferred to selection media containing3 mg/L Bialaphos until embryogenic isolates were obtained. Recoveredisolates were bulked up by transferring to fresh selection medium at2-week intervals for regeneration and further analysis. Those skilled inthe art of maize transformation will understand that other methods ofselection of transformed plants are available when other plantexpressible selectable marker genes (e.g. herbicide tolerance genes) areused.

Three constructs of IRDIG35563 contained in plasmids pDAB134672,pDAB134673, and pDAB134674 were transformed into maize (Table 7). T0plants were regenerated and tested for their ability to control targetinsect pests.

TABLE 7 Plasmid: Description: pDAB134672 ZmUbi1-IRDIG35563.3-StPinII,ZC18, ZmUbi1- AAD1-ZmLip pDAB134673 ZmCAB-IRDIG35563.3-StPinII, ZC18,ZmUbi1- AAD1-ZmLip pDAB134674 OsUbi1-IRDIG35563.3-OsUbi1, ZC18, ZmUbi1-AAD1-ZmLip

A 1.0×0.5 inch square leaf cutting was taken in duplicate from the V3 orV4 leaf of V5 To transgenic plants. For each test, wild type plant leaftissues were sampled first to prevent cross contamination of Bt toxinson the leaf edges, followed by the transformed plants. In each bioassaytray well (32-well trays and lids, CD International, Pitman, N.J.), 1leaf cutting was placed on 2% water-agar (Fisher Scientific, Fair Lawn,N.J.) and this was replicated 2 times per insect species, per event andper construct.

Each well was infested with ten FAW neonates (24-48 hrs old) and sealedwith a plastic perforated lid to allow for air exchange. The trays wereplaced at 28° C. (16:8 hr light:dark, 40% RH) and after 3 days they weregraded for percent leaf damage. The percent leaf damage data wasanalyzed with ANOVA and mean separations with the Tukey-Kramer test whenvariances are homogenous by using JMP® Pro 9.0.1 (2010 SAS InstituteInc., Cary, N.C.). Results of FAW leaf damage in T0 maize expressingIRDIG35563 is presented in Table 8.

TABLE 8 Construct Events Mean (Avg. Dmg.) Standard Error pDAB134672 1136.3 14.8 pDAB134673 25 58 6.08 pDAB134674 17 6.48 5.38 B104 9 99.2 0.59Herculex 9 22.2 4.65

Example 11

Regeneration and seed production. For regeneration, the cultures aretransferred to “28” induction medium (MS salts and vitamins, 30 gm/Lsucrose, 5 mg/L Benzylaminopurine, 0.25 mg/L 2, 4-D, 3 mg/L Bialaphos,250 mg/L cefotaxime, 2.5 gm/L Gellan gum, pH 5.7) for 1 week underlow-light conditions (14 μEm⁻²s⁻¹) then 1 week under high-lightconditions (approximately 89 μEm⁻²s⁻¹). Tissues are subsequentlytransferred to “36” regeneration medium (same as induction medium exceptlacking plant growth regulators). Plantlets 3-5 cm in length aretransferred to glass culture tubes containing SHGA medium (Schenk andHildebrandt salts and vitamins (1972); PhytoTechnology Laboratories.,Lenexa, Kans.), 1.0 gm/L myo-inositol, 10 gm/L sucrose and 2.0 gm/LGellan gum, pH 5.8) to allow for further growth and development of theshoot and roots. Plants are transplanted to the same soil mixture asdescribed earlier herein and grown to flowering in the greenhouse.Controlled pollinations for seed production are conducted.

Example 12

Design of a plant-optimized version of the coding sequence forIRDIG35563. A DNA sequence having a plant codon bias was designed andsynthesized to produce the IRDIG35563 protein in transgenic monocot anddicot plants. A codon usage table for maize (Zea mays L.) was calculatedfrom 706 protein coding sequences (CDs) obtained from sequencesdeposited in GenBank. Codon usage tables for tobacco (Nicotiana tabacum,1268 CDs), canola (Brassica napus, 530 CDs), cotton (Gossypium hirsutum,197 CDs), and soybean (Glycine max; ca. 1000 CDs) were downloaded fromdata at the website http://www.kazusa.or.jp/codon/. A biased codon setthat comprises highly used codons common to both maize and dicotdatasets, in appropriate weighted average relative amounts, wascalculated after omitting any redundant codon used less than about 10%of total codon uses for that amino acid in either plant type. To derivea plant optimized sequence encoding the IRDIG35563 protein, codonsubstitutions to the experimentally determined IRDIG35563 DNA sequencewere made such that the resulting DNA sequence had the overall codoncomposition of the plant-optimized codon bias table. Further refinementsof the sequence were made to eliminate undesirable restriction enzymerecognition sites, potential plant intron splice sites, long runs of A/Tor C/G residues, and other motifs that might interfere with RNAstability, transcription, or translation of the coding region in plantcells. Other changes were made to introduce desired restriction enzymerecognition sites, and to eliminate long internal Open Reading Frames(frames other than +1). These changes were all made within theconstraints of retaining the plant-biased codon composition. Synthesisof the designed sequence was performed by a commercial vendor (DNA2.0,Menlo Park, Calif.). Additional guidance regarding the production ofsynthetic genes can be found in, for example, WO 97/13402 and U.S. Pat.No. 5,380,831.

A maize-optimized high GC DNA sequence encoding IRDIG35563 is given inSEQ ID NO:3. A dicot-optimized DNA sequence encoding the full lengthIRDIG35563 is disclosed as SEQ ID NO:4.

Hybridization of immobilized DNA on Southern blots with radioactivelylabeled gene-specific probes may be performed by standard methods(Sambrook et al., supra.). Radioactive isotopes used for labelingpolynucleotide probes may include 32P, 33P, 14C, or 3H. Incorporation ofradioactive isotopes into polynucleotide probe molecules may be done byany of several methods well known to those skilled in the field ofmolecular biology (See, e.g. Sambrook et al., supra.). In general,hybridization and subsequent washes may be carried out under stringentconditions that allow for detection of target sequences with homology tothe claimed toxin encoding genes. For double-stranded DNA gene probes,hybridization may be carried out overnight at 20° C. to 25° C. below theT_(m) of the DNA hybrid in 6×SSPE, 5×Denhardt's Solution, 0.1% SDS, 0.1mg/mL denatured DNA [20×SSPE is 3M NaCl, 0.2 M NaHPO₄, and 0.02M EDTA(ethylenediamine tetra-acetic acid sodium salt); 100×Denhardt's Solutionis 20 gm/L Polyvinylpyrollidone, 20 gm/L Ficoll type 400 and 20 gm/L BSA(fraction V)].

Washes may typically be carried out as follows:

-   -   a. Twice at room temperature for 15 minutes in 1×SSPE, 0.1% SDS        (low stringency wash).    -   b. Once at 20° C. below the T_(m) temperature for 15 minutes in        0.2×SSPE, 0.1% SDS (moderate stringency wash).

For oligonucleotide probes, hybridization may be carried out overnightat 10° C. to 20° C. below the T_(m) of the hybrid in 6×SSPE,5×Denhardt's solution, 0.1% SDS, 0.1 mg/mL denatured DNA. T_(m) foroligonucleotide probes may be determined by the following formula (Suggset al., 1981).T _(m)(° C.)=2(number of T/A base pairs)+4(number of G/C base pairs)

Washes may typically be carried out as follows:

-   -   a. Twice at room temperature for 15 minutes 1×SSPE, 0.1% SDS        (low stringency wash).    -   b. Once at the hybridization temperature for 15 minutes in        1×SSPE, 0.1% SDS (moderate stringency wash).

Probe molecules for hybridization and hybrid molecules formed betweenprobe and target molecules may be rendered detectable by means otherthan radioactive labeling. Such alternate methods are intended to bewithin the scope of this invention.

Example 13

Transformation of Additional Crop Species. Cotton is transformed withIRDIG35563 (with or without a chloroplast transit peptide) to providecontrol of insect pests by utilizing a method known to those of skill inthe art, for example, substantially the same techniques previouslydescribed in Example 14 of U.S. Pat. No. 7,838,733, or Example 12 of PCTInternational Patent Publication No. WO 2007/053482.

While the present disclosure may be susceptible to various modificationsand alternative forms, specific embodiments have been described by wayof example in detail herein. However, it should be understood that thepresent disclosure is not intended to be limited to the particular formsdisclosed. Rather, the present disclosure is to cover all modifications,equivalents, and alternatives falling within the scope of the presentdisclosure as defined by the following appended claims and their legalequivalents.

BIBLIOGRAPHY

-   Beltz, G. A., Jacobs, K. A., Eickbush, T. H., Cherbas, P. T.,    Kafatos, F. C. (1983) Isolation of multigene families and    determination of homologies by filter hybridization methods. In Wu,    R., Grossman, L., Moldave, K. (eds.) Methods of Enzymology, Vol. 100    Academic Press, New York pp. 266-285.-   Crickmore, N., Baum, J., Bravo, A., Lereclus, D., Narva, K.,    Sampson, K., Schnepf, E., Sun, M. and Zeigler, D. R. “Bacillus    thuringiensis toxin nomenclature” (2016)    http://www.btnomenclature.info/-   Huang, F., Rogers, L. B., Rhett, G. H. (2006) Comparative    susceptibility of European corn borer, southwestern corn borer, and    sugarcane borer (Lepidoptera: Crambidae) to Cry1Ab protein in a    commercial Bacillus thuringiensis corn hybrid. J. Econ. Entomol.    99:194-202.-   Lee, et. al. (2003) The Mode of Action of the Bacillus thuringiensis    Vegetative Insecticidal Protein Vip3A Differs from That of Cry1Ab    S-Endotoxin. Appl. Environ. Microbiol. vol. 69 no. 8 4648-4657-   Sambrook, J., Fritsch, E. F., Maniatis, T. (1989) Molecular Cloning:    A Laboratory Manual (2nd ed., Cold Spring Harbor Laboratory Press,    Plainview, N.Y.)-   Tijssen, P. (1993) Laboratory Techniques in Biochemistry and    Molecular Biology Hybridization with Nucleic Acid Probes, Part I,    Chapter 2. P. C. van der Vliet (ed.), (Elsevier, N.Y.)-   Wang, Kan. Agrobacterium Protocols. 2. 1. New Jersey: Humana    Press, 2006. Print-   Weigel, D., Glazebrook, J. (eds.) (2002) Arabidopsis: A Laboratory    Manual. Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 354    pages.-   Yu, C. G., Mullins, M. A., Warren, G. W., Koziel, M. G. and    Estruch, J. J. (1997) The Bacillus thuringiensis vegetative    insecticidal protein Vip3A lyses midgut epithelium cells of    susceptible insects. Appl. Environ. Microbiol. February 1997; 63:2    532-6.

What is claimed is:
 1. A recombinant nucleic acid construct comprisingone or more heterologous regulatory elements that drive expression of anucleic acid sequence encoding a polypeptide having insecticidalactivity and having at least 96% sequence identity to the polypeptidesequence of SEQ ID NO: 2, and one or more heterologous regulatoryelements that drive expression of a nucleic acid sequence encodingeither a further polypeptide having insecticidal activity or apolypeptide that confers resistance to an herbicide.
 2. The construct ofclaim 1 in which the nucleic acid sequence encoding the polypeptidehaving at least 96% sequence identity to the polypeptide sequence of SEQID NO: 2 is chosen from the group consisting of SEQ ID NO:1, SEQ IDNO:3, and SEQ ID NO:4.
 3. A plant or plant part comprising a nucleicacid construct of claim
 1. 4. A plant or plant part comprising a nucleicacid construct of claim
 2. 5. The plant part of claim 3 wherein theplant part is a seed.
 6. The plant part of claim 4 wherein the plantpart is a seed.
 7. The plant or plant part of claim 3, wherein thenucleic acid construct encodes a chimeric toxin having insecticidalactivity against one or more insects selected from Spodoptera exigua(Beet armyworm, BAW), Spodoptera eridania (Southern armyworm, SAW),Spodoptera frupperda (Fall armyworm, FAW), resistant Spodopterafrupperda (Fall armyworm, FAW), Helicoverpa zea (Corn earworm, CEW),Pseudoplusia includens (Soybean looper, SBL), Anticarsia gemmatalis(Velvetbean caterpillar, VBC), Heliothis virescens (Tobacco budworm,TBW), and Hehcoverpa armigera (Cotton bollworm, CBW).
 8. A plant orplant part of claim 4 wherein the nucleic acid construct encodes achimeric toxin having insecticidal activity against one or more insectsselected from Spodoptera exigua (Beet armyworm, BAW), Spodopteraeridania (Southern armyworm, SAW), Spodoptera frupperda (Fall armyworm,FAW), resistant Spodoptera frupperda (rFall armyworm, rFAW), Helicoverpazea (Corn earworm, CEW), Pseudoplusia includens (Soybean looper, SBL),Anticarsia gemmatalis (Velvetbean caterpillar, VBC), Heliothis virescens(Tobacco budworm, TBW), and Hehcoverpa armigera (Cotton bollworm, CBW).9. A method for producing an insect resistant or insect tolerant plantcomprising breeding a non-transgenic plant with a transgenic plantcomprising a construct of claim 1 stably incorporated into the genome ofthe plant and selecting progeny containing the DNA construct of claim 1.